Introduction

Acute myeloid leukemia (AML), a malignancy stemming from alterations in myeloid hematopoietic stem cells, results in abnormal shifts in the ratio of primitive to naive myeloid cells. AML is the most prevalent justification for allogeneic hematopoietic stem cell transplantation (allo-HSCT). The main mechanism of allo-HSCT in treating AML is the graft-versus-leukemia (GVL) effect, mediated by transplanted donor T cells, which facilitate the reconstitution of the recipient’s immune system. However, while the GVL effect is beneficial, donor T cells also contribute to the life-threatening complication of graft-versus-host disease (GVHD),1 2 a serious immune disease resulting in multiorgan damage and significantly impacting transplantation outcome and postoperative survival.3 4

The pathological process of acute GVHD unfolds in three primary stages.5 Initially, pre-transplantation chemotherapy or irradiation leads to upper barrier dysfunction, creating and sustaining an inflammatory environment. This results in an increased secretion of pro-inflammatory cytokines that stimulate the activation of recipient antigen-presenting cells (APCs). Subsequently, donor T cells interact with recipient APCs, triggering donor T-cell activation. In the final stage, these activated T cells migrate to target tissues like the skin, liver, and intestines, damaging recipient tissues and causing multiorgan failure. An intricate balance exists between GVHD and GVL, complicating the body’s ability to entirely avoid immune rejection and leukemia relapse post-HSCT.6 The manipulation of synergistic signaling on T-cell affects both effector and regulatory T cells, leading to either immune tolerance or overactivation.7

A promising emerging technology in cell surface engineering is single-cell nanoencapsulation, with numerous biomedical applications.8–11 By encapsulating individual mammalian cells with semipermeable and biocompatible materials, the resulting vesicle shell functions as an immunoisolating barrier. However, the technology has seen limited application in nanoencapsulating individual immune cell, requiring mild operating conditions to safeguard the fragile membranes of immune cells and ensure the preservation of their functions.12

Layer-by-layer (LbL) assembly of polyelectrolytes on cell surfaces is a promising strategy for cellular nanoencapsulation, allowing precise control over the capsule wall thickness and LbL self-assembly properties.13 14 The prepared microcapsules, with small particle size and smooth surfaces, feature a semipermeable membrane permitting the passage of oxygen, nutrients, metabolites, and small-molecule proteins, while preventing larger, immunologically active macromolecules from passing through.14 15

Our research employed polycationic aminated A-type gelatin and polyanionic alginate to form thin nanomembranes on T-cell surfaces using the LbL self-assembly technique. Observations confirmed that the nanoencapsulation process did not affect the functions of the encapsulated T cells, including viability, proliferation, material degradation time, and cytokine secretion levels. Encapsulated donor T cells mitigated the onset and progression of GVHD in the early stages of transplantation by diminishing co-stimulatory signals with recipient APCs through immune isolation. Furthermore, donor T cells resumed their tumor-killing effect on AML cells after nanocapsule shell degradation, employing the time difference to inhibit GVHD while maintaining the GVL effect. This novel technique has been authorized by China National Invention Patent (No.202111025332.1).

Materials and methodsReagents

The following reagents were obtained from Sigma-Aldrich (USA): Gelatin (G2625), alginate (A2158), fluorescein 5 (6)-isothiocyanate (F3651), busulfan (B2635), cyclophosphamide (PHR1404), FITC-dextran (BCCC2378). The following reagents were obtained from Macklin (China): hexamethyldisilane (H810965), 2-(N-morpholino) ethanesulfonic (M813439), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (N808856). N-hydroxysulfosuccinimide was purchased from Solarbio (China). Z-IETD-FMK (210344-98-2) was purchased from MCE (America). was purchased from MCE (America).

The following antibodies were obtained from eBioscience (USA): APC-conjugated anti-mouse LFA-1 (17-0111-82), APC-conjugated anti-mouse CD154 (17-1541-81), PE-conjugated anti-mouse CD80 (12-0801-81). The following antibodies were obtained from BioLegend (USA): PE-Cy7-conjugated anti-mouse H-2Kb (116520), AF647-conjugated anti-mouse H-2Kd (116612), BV421-conjugated anti-mouse CD3 (100228), BV421-conjugated anti-mouse CD11c (117329), FITC-conjugated anti-mouse I-A/I-E (107616), APC-conjugated anti-mouse CD54 (116119), PE-conjugated anti-mouse CD275 (107405), BV421-conjugated anti-mouse CD45.1 (110731), BV510-conjugated anti-mouse CD45.2 (109838), PE-Cy5.5-conjugated anti-mouse CD25 (101911), AF647-conjugated anti-mouse Foxp3 (126407), BV421-conjugated anti-mouse IL-17A (506925), APC-conjugated anti-mouse perforin (154403), BV421-conjugated anti-mouse granzyme B (396413). Purified anti-mouse CD3 Antibody (100340), purified anti-mouse CD28 Antibody (102116). The following reagents were obtained from BD Biosciences (USA): FITC-conjugated anti-mouse CD4 (553650), PE-conjugated anti-mouse CD8 (553032), APC-conjugated anti-mouse CD278 (565883), PE-conjugated anti-mouse CD40 (561846), PE-conjugated anti-mouse CD3 (553063), PE-conjugated anti-mouse CD19 (553786), PE-Cy5.5-conjugated anti-mouse Lineage Cocktail (561317), FITC-conjugated anti-human CD68 (562111), PE-conjugated anti-human CD86 (560957), BV421-conjugated anti-human CD206 (566281), BB515-conjugated anti-human CD40 (585927), FITC-conjugated anti-human CD40L (566628), FITC-conjugated anti-human CD28 (535721), PE-conjugated anti-human CD3 (561803).

The following antibodies were obtained from ZEN-BIOSCIENCE (China): Bcl2 rabbit pAb (381702), bax rabbit pAb (380709). The following antibodies were obtained from Bioss (China): Fas ligand rabbit pAb (bs-0216R), rabbit anti-CD95/FAS antibody (bs-6477R), caspase-3 p17 subunit rabbit pAb (bs-20364R), goat anti-rabbit IgG(H+L) (E-AB-1003) was purchased from Elabscience (China).

Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and murine interleukin (IL-4) were purchased from PeproTech (USA).

APC-conjugated anti-mouse CD28 (130-111-973) and Anti-PE MicroBeads (130-048-801) were purchased from Miltenyi (Germany). Mice ELISA Assay Kits were purchased from Fcmacs Biotech (China), FITC annexin V Apoptosis Detection Kit I (556547) was purchased from BD Biosciences (USA). Cell Counting Kit-8 (C0037) was purchased from Beyotime. CFSE Cell Proliferation Assay Kit was purchased from eBioscience (65-0850-84) (USA). Cell Counting Kit-8 (C0037) was purchased from Beyotime. CFSE Cell Proliferation Assay Kit was purchased from eBioscience (65-0850-84).

Cat and PRID numbers for all key resources are organized in table 1.

Table 1

Key resource table

Mice

Two congenic strains of donor mice (female, 8–10 weeks) were used: C57BL/6 mice with genetic background Ly-5.1 (donor bone marrow cells (BMCs) were extracted) purchased from Nanjing Junke Bioengineering (China); wild-type C57BL/6 mice with genetic background Ly-5.2 (donor T cells were extracted), and recipient BALB/c (H2d) mice were purchased from Beijing Spefo Biotechnology (China).

Mice were housed in the Specific Pathogen Free Animal Laboratory, Institute of Clinical Pharmacology, Anhui Medical University. All animal experiments were approved by the Animal Experimentation Ethics Committee of the Institute of Clinical Pharmacology, Anhui Medical University (Approval No. PZ-2021–026), and all experiments were conducted in accordance with the approved standards and procedures.

Cell culture

The BALB/c (H2d) myeloid leukemia WEHI3B cell line was purchased from Nanjing SHRBIO Biological (China) used in this study. The WEHI-3B cells were further modified by transducing with a luciferase gene (thereafter WEHI-3B-luc), which allowed for non-invasive visualization of tumor progression. Both modified and unmodified WEHI-3B cells were cultured in RPMI 1640 (Gibco, USA) supplemented with 10% heat-inactivated FBS (Wisent, Canada), 100 unit/mL penicillin (Life Technologies), 100 µg/mL streptomycin (Life Technologies), and 50 µm 2-mercaptoethanol (Sigma, USA).

The C57BL/6J (H2b) myeloid leukemia C1498 cell line was purchased from Wuhan SUNNCELL Biological (China) used in this study. C1498 cells were cultured in DMEM (Gibco, USA) supplemented with 10% heat-inactivated FBS (Wisent, Canada), 100 unit/mL penicillin (Life Technologies), 100 µg/mL streptomycin (Life Technologies), and 50 µm 2-mercaptoethanol (Sigma, USA).

Preparation of cationic gelatin and fluorescence labeled alginate

Cationic gelatin is produced in the following steps as illustrated in online supplemental figure S1. A mixture of 2 mL of ethylenediamine and 1.0 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was added to 50 mL of 0.1 M phosphate-buffered saline (PBS) containing 1.0 g of gelatin type A. The reaction was carried out at 37°C for 18 hours. The reaction was gently stirred at 37°C for 18 hours. The reaction mixture was then dialyzed with purified water for 48 hours. Finally, the dialysate was freeze-dried to obtain cationic gelatin.

Fluorescein isothiocyanate (FITC)-alginate was produced by the following steps. 9 mM EDC and 9 mM Sulfo-NHS were prepared using 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Alginate (1.8%, w/v) was dissolved with MES, the pH was adjusted to 4.7, followed by slow addition of EDC solution for 15-min reaction, the pH was adjusted to 7.4 using PBS buffer, and then Sulfo-NHS solution was added slowly. After stirring for 2 hours at room temperature, 2 Mm FITC was added and stirred for 18 hours at room temperature away from light. The final solution was dialyzed with 1 M NaCl solution and distilled water, respectively, for 24 hours protected from light, and the solution in the dialysis bag was freeze-dried to obtain FITC-labeled alginate.

LbL single T-cell encapsulation

First, 2×106 T cells were centrifuged to remove the medium within a 15 mL centrifuge tube. Next, 1 mL of 0.2% gelatin solution was added to the centrifuge tube and placed in a constant temperature shaker incubator for shaking and mixing (200 rpm×3 min), and then the incubation was continued for another 10 min, with slight oscillation every 2 min. Then, the tube was centrifuged at 2,000 rpm for 5 min, after which, the supernatant was discarded. Cells were washed by adding 5 mL Dulbecco's phosphate-buffered saline (DPBS), then the tube was centrifuged again, and the supernatant was discarded. After the cells were washed for a second time, 1 mL of 0.25% alginate was incubated with the cells for 10 min. The process was repeated several times, and a layer of gelatin and alginate was encapsulated to complete the four-layered LbL encapsulation model of T cells.

Cell viability test with Hoechst/PI staining

The number of T cells in the normal and LbL self-assembly groups was controlled at 1×106. Cells were washed twice with PBS. Cells were resuspended with 500 µL of staining buffer and then 5 µL of Hoechst 33,342 staining solution A (Solarbio, China) was added. The solution was gently mixed and incubated for 10 min at 4°C protected from light before adding 5 µL of propidium iodide (PI) staining solution B. After incubation in the same conditions the cells were washed with PBS and resuspended. Finally, the results were finally examined by flow cytometry or fluorescence microscopy.

Characterization of encapsulated T cell

T cells at the density of 2×106 were nanoencapsulated as described above. The same amount of untreated T cells, T cells encapsulated with gelatin, gelatin/alginate, and (gelatin)2/alginate, (gelatin)2/(alginate)2 was taken, respectively. Then, the zeta potential was determined by the instrument (Malvern, UK).

To visualize the porous film conformally encapsulated over T cells, the FITC-labeled alginate was used for encapsulation with the same method. The thickness of the conformal film was examined using a confocal microscope (Leica SP8). We randomly selected five different locations for each cell to measure thickness using ImageJ (Bethesda, USA) and measured a total of five encapsulated T cells to obtain the mean and SE of membrane thickness.

T cells and encapsulated T cells were fixed in 5% glutaraldehyde (0.1 M PBS dilution) for 30 min, respectively. Samples were dehydrated with gradient alcohol for 10 min each. Next, a solution of 1:2, 2:1 (hexamethyldisilazane (HMDS): alcohol), and 100% HMDS was transferred to the samples to dry them, respectively. Finally, the samples were sprayed with gold and observed with SEM (ZEISS Gemini). For co-culture experiments, the area and diameter of cells were quantified using ImageJ software.

Cytokine measurement by ELISA

A total of 4×105 encapsulated T cells were activated by Ultra-LEAF Purified anti-mouse CD3/CD28 (3 µg/mL) in 96-well plates for 48 or 96 hours, and then the supernatant was collected. Levels of IL-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were assayed using ELISA kits according to the manufacturer’s instructions.

After recipient mice were sacrificed, peritoneal macrophage culture supernatants after incubating in well plates at a density of 2×105 /mL for 24 hours were collected. Peripheral blood serum samples were collected. Levels of IL-6, IL-10, IFN-γ, and C-X-C motif chemokine ligand 10 (CXCL10) were measured using ELISA kits according to the manufacturer’s instructions.

Bone marrow-derived dendritic cell induction mixed lymphocyte culture

BALB/c mice were euthanized and sterilized with 75% ethanol for 3 min. Bone marrow-derived mononuclear cells (BMDCs) were extracted under aseptic conditions and then dispensed into 24-well plates at a density of 1×106 cells/mL/well. Subsequently, GM-CSF and IL-4 were added to the medium to a final concentration of 20 ng/mL and 10 ng/mL, respectively. The cells were incubated in an incubator containing 5% CO2 at 37°C. The medium was changed after 12 hours to remove unattached cells and cell debris, and then GM-CSF and IL-4 were supplemented to the fresh medium. On day 7, cells were collected in semi-suspension by gently transferred to the plate to collect semi-suspended cells and loosely attached cells. Cells were inoculated into 6-well plates and incubated with lipopolysaccharide (LPS) for 24 hours to obtain BMDCs.

CD3+T cells (1×105) were co-cultured with allogeneic dendritic cells (DCs) (1×104) for 2–4 days in 96-well plates in triplicates in 200 µL complete medium per well. For DC-independent T-cell proliferation assays, CD3+T cells (1×105) were activated by Ultra-LEAF Purified anti-mouse CD3/CD28 (3 µg/mL) for 48 hours. The experiments were performed in Roswell Park Memorial Institute (RPMI) 1640 with IL-2 (20 ng/mL). T-cell proliferation was assessed by carboxyfluorescein succinimidyl ester (CFSE) dilution as previously described.

Observation and detection of immune synapses

T cells were co-cultured with matured DCs grown on polylysine slides for 6 hours. DCs were sensitized by adding ovalbumin OVA for 4 hours before co-culture.

Cells were collected and fixed by adding glutaraldehyde containing 2.5% glutaraldehyde. They were resuspended with PBS buffer containing 8% sucrose, washed twice with pre-cooled PBS, and incubated at 4°C overnight. An equal amount of 1% agarose gel with 0.5 M sorbitol in ultralight water was subsequently added. A secondary fixation in 2% osmium acid was then performed for 2 hours. Dehydration was performed using graded ethanol and then embedded in epoxy resin. After drying in an oven at 60°C for 48 hours, thin sections (90±10 nm) were cut with a Leica EM UC7 ultrathin sectioning machine and mounted on a copper mesh. The sections were stained with 5% aqueous uranyl acetate and 0.2% lead citrate, left to dry, and then observed and photographed under a 120 kV transmission electron microscope (Thermo Fisher).

For confocal micrographs. Cells were fixed with 4% paraformaldehyde. Cells were then permeabilized with PBS containing 0.1% Triton X-100, blocked with 1% bovine serum albumin (BSA)/PBS for 1 hour, rinsed with PBS, and incubated with anti-CD3, major histocompatibility complex (MHC)-II antibody at 4°C overnight. After washing, secondary antibodies were added and incubated at room temperature away from light for 1 hour. For actin staining, cells were incubated with AF657-Ghost Pen Cyclic Peptide in PBS for 40 min at room temperature. Labeled microvilli and actin were observed in the cells. 4',6-diamidino-2-phenylindole (DAPI) was then added to continue the incubation for 20 min. Cells were imaged using a 64×oil immersion objective and an SP8 laser scanning confocal microscope (Leica).

Leukemia and GVHD models

This study involves BALB/c mice divided into four distinct groups to explore the implications of various grafts. NC group, BMCs group, BMCs+T cell transplant group, and BMCs+encapsulated donor T-cell transplants. Prior to transplantation, the treated groups receive busulfan (25 mg/kg/days) and cyclophosphamide (125 mg/kg/days), acting as chemotherapeutic and immunosuppressive agents to aid the transplant process. In addition, Luc-WEHI-3B cells transduced with a lentivirus were injected via tail vein. On the day of transplantation, these groups receive BMCs (5×106 cells per mouse) and T cells (2×106 cells per mouse) derived from donor mice, in line with their group assignment. The ratio of transplanted cells is approximately 2.5:1 for BMCs to T cells.

Assessment of GVHD

The recipient mice were marked with ear punches, and their individual weights were recorded on the first day, with subsequent recordings made every 3 days thereafter. Concurrently, these mice were subjected to daily monitoring for clinical indications of GVHD and survival rates. The clinical score of GVHD was assessed by a scoring system described in table 2 that incorporates five physical parameters: weight loss, posture (hunching), activity, fur texture, diarrhea, and skin integrity. Each mouse was assessed and allocated a grade ranging from 0 to 2 for each criterion. Subsequently, a cumulative clinical index score was generated through the summation of these six individual criterion scores (maximum index=12, table 2). The pathological scores of H&E-stained sections of the liver, spleen, skin and colon were performed according to the scoring criteria of tables 3–6.

Table 2

Assessment of clinical graft-versus-host disease in transplanted animals

Table 6

Assessment of H&E-stained images of the colon from transplanted animals

Table 5

Assessment of H&E-stained images of skin from transplanted animals

Table 4

Assessment of H&E-stained images of spleen from transplanted animals

Table 3

Assessment of H&E-stained images of liver from transplanted animals

In vivo bioluminescence imaging

The IVIS Spectrum whole-animal imaging system (PerkinElmer, USA) was employed for the non-invasive live imaging of tumor cells. Mice were anesthetized with isoflurane (RWD, China), followed by an intraperitoneal injection of the firefly luciferin substrate (diluted to 5 mg/mL in PBS) at a dosage of 150 mg/kg body weight. The IVIS imaging was initiated 15 min post substrate injection. The whole-body bioluminescent signal intensity was documented on a weekly basis. For the purpose of material tracking, chemiluminescence was calibrated to an output of 480 nm, enabling the detection of FITC fluorescence.

Cell proliferation assay

After DC maturation, DCs and T cells (or encapsulated T cells) were co-cultured at a 1:10 ratio at 1×106 T cells/mL in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). IL-2 (20 ng/mL) was added to some cultures. CFSE-labeled T cells were cultured alone or in the presence of cytokine-matured DCs in RPMI 1640 in 24-well plates. Flow cytometry was performed on day 2 or 4 of co-culture to assess the proliferation of T cells.

Apoptosis detection

WEHI-3B (or C1498) and T cells (or encapsulated T cells) were co-cultured at a 1:10 ratio at 1×106 T cells/mL in RPMI 1640 supplemented with 10% FBS. WEHI-3B cells were collected and processed according to the manufacturer’s instructions for the FITC annexin V Apoptosis Detection Kit. Flow cytometry was performed at times 24 hours, 48 hours or 96 hours of co-culture to detect the apoptosis of tumor cells.

Cell Counting Kit-8 assay

WEHI-3B or C1498 and T cells (or encapsulated T cells) were co-cultured as above in 24-well plates or Transwell chamber. WEHI-3B or C1498 cells were transferred to 96-well plates at 6 hours, 24 hours, 48 hours, and 72 hours and incubated for 1 hour with Cell Counting Kit-8 reagent. Optical density was detected by Multifunctional Enzyme Marker (Tecan, Switzerland).

High content analysis

300 µL of WEHI-3B (1×105 cells/well) containing 10% RPMI 1640 medium was added to the 24-well plate for 5 hours, and then 200 µL of encapsulated and non-encapsulated T cells (1×106 cells/well) were evenly seeded into the well plate. The cells were incubated in 5% CO2 incubator for 24 hours, 48 hours, and 72 hours, respectively. At the end of each incubation time, the supernatant was discarded, and the cells were washed with PBS by gently shaking, fixed with precooled 4% paraformaldehyde for 30 min, and washed three times with PBS after the end. DAPI was added to stain the nucleus for 5 min, then PBS was slowly added along the wall and washed three times with slight shaking. Finally, photos were taken with the high-content imaging system.

Western blot

Proteins from tumor cells or T cells were extracted from a 48 hours co-culture system, separated on 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gels, and transferred to polyvinylidene difluoride membrane (Millipore Corporation, Billerica, Massachusetts, USA). The membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% non-fat milk at 37°C for 2 hours, followed by incubation with a primary antibody against added, and the mixture was incubated overnight at 4°C. The membranes were washed with TBST three times; the secondary antibody was added, incubated at room temperature for 2 hours, washed three times with TBST, and developed. Image software was used to analyze the gray value of each band, and the control protein was used to normalize the gray values of the target protein for statistical analysis. The membranes were blocked in TBST and 5% non-fat milk at 37°C for 2 hours, followed by incubation with a primary antibody against were added and the mixture was incubated overnight at 4°C. The membranes were washed with TBST three times; the secondary antibody was added, incubated at room temperature for 2 hours, washed three times with TBST, and developed. Image software was used to analyze the gray value of each band, and the control protein was used to normalize the gray values of the target protein for statistical analysis.

Isolation and sorting of PBMCs derived T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy peripheral blood using a human lymphocyte separation medium. Isolation and sorting of PBMC-derived T cells and encapsulation of single cell.

Induction of macrophages in PBMCs and detection of the expression of macrophage and T cells co-stimulatory molecules in the co-culture system

Isolated PBMCs were cultured for 4 hours, non-adherent cells in the supernatant were washed away, 10% FBS 1640 and 100 ng/mL M-CSF were added to the culture. 500 ng/mL LPS was added after 6 days of culture. On day 7, the cells were removed, the supernatant was discarded, and the cells were carefully scraped. The cells were washed with PBS, the supernatant was discarded and resuspended in 100 µL PBS for cell suspensions and counted.

Encapsulated and non-encapsulated human T cells were co-cultured in 24-well plates at a cell ratio of 10:1 to macrophages for 24 hours. CD3+lymphocyte function-associated antigen-1 (LFA-1)+ on T cells and the expression levels of co-stimulatory molecules CD28:CD86, CD40L:CD40 were measured by flow cytometry.

Flow cytometry analyses

Murine splenocytes or blood were isolated and T cells stained with PE-Cy7-conjugated H2Kb, BV421 or PE-conjugated CD3, FITC-conjugated CD4, APC-conjugated CD8, CD28, CD40L, ICOS according to the manufacturer’s instructions. For DCs, the cells stained with APC-conjugated H-2Kd, PE-Cy5.5 conjugated anti-mouse Lineage Cocktail, BV421 conjugated CD11c, FITC conjugated MHC-II, PE-conjugated ICAM-1, CD80, CD40, ICOSL (gating strategies are represented in online supplemental figure S9). Perforin, granzyme B, and T helper cell 17 (Th17)/regulatory T cell (Treg) cell subsets were labeled in T cells. T cells were isolated from a co-culture system in vitro. T cells stained with APC-conjugated perforin, BV421-conjugated granzyme B, FITC-conjugated CD4 and PE-conjugated CD8.

Cells were analyzed using FACSCanto II (BD Biosciences) and FlowJo software (TreeStar).

Statistics

Survival was ascertained by Kaplan-Meier log-rank analyses. Statistical analyses were performed with the Student’s t-test, analysis of variance, or log-rank test as appropriate. Prism V.8 software was used for statistical analysis. Values of p<0.05 were considered statistically significant.

ResultsFunctional characteristics of individual T cells after conformal nanoencapsulation with natural degradable materials

We achieved the nanoencapsulation of individual T cells using an LbL assembly approach, leveraging the electrostatic properties of T cells with negatively charged surfaces. This was accomplished by repeatedly immersing these T cells into a cationic gelatin solution and anionic alginate, resulting in four layers of encapsulated microcapsules (figure 1A). We observed alternating positive and negative zeta potentials on the surface of T cells during encapsulation, with the potential ultimately resembling the initial state prior to the encapsulation figure 1B). To monitor and quantify the encapsulation process, we used FITC fluorescein attached to alginate, which enabled us to track alterations in the microcapsules (figure 1C). This was followed by the encapsulation of isolated and sorted T cells. The encapsulation efficiency, after defining the optimal concentration ratio (online supplemental information, online supplemental figure S2) and incubation time (online supplemental figure S2, online supplemental figure S3A) of the selected materials, was found to be approximately 90%, after a 10 min incubation period with mixed oscillations (figure 1D). This was determined using flow cytometry. Confocal imaging was employed to visualize the fluorescence expressed by the outermost layer of alginate in the conformal film of encapsulated T cells (figure 1C). Scanning electron microscopy revealed a rich display of microvilli on the topography of non-encapsulated T cells, contrasting with the streamlined and spheroidal surface of encapsulated T cells. The diameters of non-encapsulated and encapsulated T cells were measured to be 6.28±0.20 µm and 7.04±0.17 µm, respectively, indicating that the gelatin and alginate encasement formed a conformal film around 400 nm in size (figure 1E). The viability and survival of T cells encapsulated via LbL assembly were comparable to non-encapsulated T cells within 6 hours post-assembly (online supplemental information, online supplemental figure S3B,C). Furthermore, we found a similar proliferative response in both encapsulated and non-encapsulated T cells after exposure to CD3/CD28 and IL-2 stimulation (figure 1F,G). The secretion levels of TNF-α, IL-2, and IFN-γ by both groups of cells showed no significant difference at 48 hours and 96 hours post-activation (figure 1H–J). Our observations also suggest that single-cell encapsulation had no impact on the binding capacity of T cells to anti-CD3 (figure 1K,L).

Figure 1

Successful conformal nanoencapsulation of T cells and preservation of original cell functions. (A) Illustration of T-cell encapsulation progression. (B) Depiction of zeta potential changes in T cells throughout the layer-by-layer (LbL) encapsulation process. (C) Absorption peak plots of both alginate and FITC-alginate at 480 nm are presented, accompanied by FITC fluorescence images of the encapsulated T cell’s outer layer. (D) Representative flow scatter plots, demonstrating the encapsulation efficiency achieved when employing a combination of 0.2% gelatin and 0.25% alginate. (E) Comparative scanning electron microscopy images of non-encapsulated and encapsulated T cells are displayed, supplemented by differential interference contrast images. Quantification of cell diameters was executed using ImageJ software. (F–G) Following 48 hours of purified CD3/CD28 antibody-stimulated proliferation, representative flow peak plots of CFSE for both encapsulated and non-encapsulated T cells are exhibited. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. (H–J) The secretion levels of TNF-α, IL-2, and IFN-γ by T cells at 48 hours and 96 hours post-activation by CD3/CD28 antibody were detected by ELISA. (K–L) Comparison of the Anti-CD3 binding capacity between non-encapsulated and encapsulated T cells. All data are represented as mean values±SE, results of at least three (G–J) or five (B, L) repeat experiments each with three samples. *p<0.05, **p<0.01. APC, antigen-presenting cell; CFSE, carboxyfluorescein succinimidyl ester; DPBS, Dulbecco's phosphate-buffered saline; FITC, fluorescein isothiocyanate; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Encapsulated T cells reduce GVHD severity and enhance survival of mice with GVHD

To explore the therapeutic impact of encapsulated T cells on GVHD mice, the recipient mice underwent a pretreatment regimen involving busulfan and cyclophosphamide (figure 2A). Following this regimen, the peripheral blood levels in the recipient mice decreased significantly to approximately 0–1×109/L. We also observed a substantial reduction in the ratio of leukocytes, neutrophils, and lymphocytes compared with levels before pretreatment (figure 2B,C).

Figure 2

Encapsulated T cells combined with BMCs transplantation inhibited the development of GVHD in recipient mice. (A) Female BALB/c recipient mice were administered 0.4 mg of busulfan and 2 mg of cyclophosphamide intraperitoneally per 20 g mouse, starting from day −7 prior to BMT. In the allogeneic hematopoietic stem cell transplantation procedure, BMCs and splenic T cells harvested from H2-b C57BL/6 mice were introduced via tail vein injection. (B–C) Analyses of blood samples were conducted on the recipient mice before and after the chemical pretreatment conditioning regimen. The analyses included quantification of various cell types such as leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. (D) On day 7, FITC-alginate encapsulated T cells were observed, and FITC fluorescence images of the recipient BALB/c mice were captured. (E) Mice in the non-encapsulated control group developed GVHD, exhibiting symptoms such as decreased mobility, depression, arched backs, hair loss, gastrointestinal hemorrhage, and skin flaking. (F–H) Over a 60-day period, observations were recorded detailing the changes in body weight, clinical scores, and survival rates of the mice in each group. (I–L) Flow scatter plots and statistical analyses were produced to determine the numbers of CD3+, CD4+, and CD8+T cell subsets in the peripheral blood of mice in both the encapsulated and non-encapsulated groups. (M–O) Representative flow scatter plots were generated to illustrate the populations of CD3+LFA-1+ T cells, CD11c+MHC-II+ cells, and CD11c+ICAM1+ cells in the spleens of mice across different groups. (P–R) Statistical analyses of the proportions of CD3+LFA-1+ T cells, CD11c+MHC-II+ cells, and CD11c+ICAM1+ cells in the spleens of mice in each group were executed. Pooled data from three independent experiments each with five or seven recipients. Survival (F) Kaplan-Meier curve), clinical score (G) weight (H) from two or three independent experiments, each with seven mice per group, are shown. Mean value±SEM; *p<0.05, ***p<0.001, non-encap versus encap; #p<0.05, ##p<0.01, ###p<0.001, non-encap versus BMCs. BMC, bone marrow cell; FITC, fluorescein isothiocyanate; GVHD, graft-versus-host disease; LFA-1, lymphocyte function-associated antigen-1; MHC, major histocompatibility complex.

Before initiating transplantation, we conducted a safety evaluation of the encapsulated T cells. Hemolysis experiments on type A gelatin, modified cationic gelatin, and alginate revealed no significant hemolysis with any of the materials used (online supplemental information, online supplemental figure S4). Alginate and gelatin were separately injected into recipient mice via the tail vein at twice the encapsulated concentration, with the PBS-injected group serving as the control. On the 14th day, we collected peripheral blood serum from the recipient mice and evaluated biochemical indices such as total protein, albumin, globulin, glutamate aminotransferase, total bilirubin, glutamine alanine aminotransferase, and urea. The results showed that cationic gelatin and alginate did not significantly impact these indices compared with the PBS control (online supplemental information, online supplemental figure S5).

We established a GVHD mouse model using BMCs combined with donor T-cell transplantation. Fluorescent labeling allowed us to observe encapsulated T cells entering the recipient mice circulation and distributing throughout the body within 7 days (figure 2D). In contrast to recipients receiving non-encapsulated T-cell transplants, who displayed signs of GVHD such as a hunched back, hair loss, gastrointestinal bleeding, and skin peeling (figure 2E), recipients of encapsulated T-cell transplants exhibited significantly lower GVHD clinical scores and extended survival times (figure 2F,G). Encapsulated T-cell treatment also mitigated weight loss in recipient mice. Despite an initial decrease in the body weights of the mice in the BMCs group and the encapsulated T-cell group, weights progressively increased from the 20th day and returned to pre-transplantation levels by the 45th day (figure 2H).

On near-death euthanasia, analysis of the mice revealed no significant difference in the proportion of peripheral blood CD3+T cells and CD8+cytotoxic T Lymphocyte (CTL) expression within the encapsulated T-cell group. Nevertheless, a noticeable decrease in the CD4/CD8 ratio was observed compared with the group with non-encapsulated donor T cells (figure 2I–L).

LFA-1 is a mechanosensitive adhesion receptor crucial for T-cell migration, differentiation, and effector functions. It achieves this via actin dynamics. Blocking the ICAM-1/LFA-1 interaction can inhibit T-cell activation in autoimmune diseases and organ transplantation.16–18 In this study, we fluorescently labeled CD3+T cells and DCs within the spleens of recipient mice. Our observations revealed that the expression levels of CD3+LFA-1+, CD11c+MHC-II+, and CD11c+ICAM-1+ in the spleens of mice receiving non-encapsulated T cells were markedly elevated compared with those in the encapsulated T-cell recipient group. However, no significant differences were observed in these results between the encapsulated group and the normal group (figure 2M–R).

Nanoencapsulation suppresses the expression of co-stimulatory molecules between donor T cells and recipient APCs and affects the formation of immune synapses

In order to probe the impact of single-cell nanoencapsulation on the signaling between donor T cells and recipient APCs, we matured DCs from the bone marrow of BALB/c mice (online supplemental information, online supplemental figure S6). We then co-cultured these mature DCs with donor T cells sourced from C57BL/6J mice, tracking their proliferation by labeling the T cells with CFSE fluorescent dye. Notably, the proliferation ratio of encapsulated T cells at the 48-hour mark was lower than that of the non-encapsulated group. Though the proliferation rate increased after 96 hours to 13.4%, it remained significantly lower than the non-encapsulated group’s proliferation rate of 53% (figure 3A,B). Flow cytometry analysis of the cells collected after 48 hours of co-culture revealed that the expression of CD28, ICOS, and CD40L on T cells in the encapsulated group was significantly lower than in the non-encapsulated group (figure 3C,E). However, the corresponding co-stimulatory molecules, CD80, ICOSL, and CD40, exhibited no significant differences in DCs (figure 3D,F).

Figure 3

Single-cell nanoencapsulation reduced the expression of co-stimulatory molecules between donor T cells and recipient antigen-presenting cells and affected the formation of immune synapses. Mature DCs were co-cultured with encapsulated or non-encapsulated donor T cells to activate unidirectional mixed lymphocyte responses. (A–B) Proliferation of CFSE-labeled T cells was monitored at 48 hours and 96 hours. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. (C–F) Representative flow cytometry histograms and associated statistical analysis of the co-stimulatory molecules CD28, ICOS, and CD40L on T cells and CD80, ICOSL, and CD40 on DCs. (G–K) DCs were activated, sensitized with OVA antigen, and co-cultured with either encapsulated or non-encapsulated donor T cells for 6 hours. Cells within this co-culture system were then collected for further analysis. (G–H) Imaging flow cytometry results comparing the encapsulated group to the non-encapsulated group. (I) Representative immunofluorescence images of T cells co-cultured with DCs in both the encapsulated and non-encapsulated groups. (J–K) Scanning electron microscopy and TEM images of T cells co-cultured with DCs in both the encapsulated and non-encapsulated groups. Mean value±SEM, results of at least five repeat experiments each with three samples. *p<0.05, **p<0.01, ***p<0.01. CFSE, carboxyfluorescein succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; DC, dendritic cell; MHC, major histocompatibility complex; TEM, transmission electron microscopy.

After 6 hours of co-culture, we detected the cytoskeletal protein F-actin, which can be tightly bound by phalloidin. We observed that the expression of F-actin on cells in the encapsulated group was lower than that in the non-encapsulated group (figure 3G,H). Additionally, the formation of the F-actin ring (red fluorescence) on the encapsulated T cells appeared to be attenuated. The discernible distance observed between encapsulated T cells and DCs suggests a failure to establish an evident immune synapse (figure 3I).

Scanning electron microscopy and transmission electron microscopy images provided a more detailed visualization of the effect of nanoencapsulation on the formation of immune synapses. Compared with the non-encapsulated group, the surface texture of encapsulated T cells was relatively regular, featuring embedded microvilli. DCs co-cultured with encapsulated T cells had shorter diameters than activated DCs, and the pseudopods were not fully extended, (figure 3J,K, online supplemental information, online supplemental figure S7A,B). In addition, the number of mitochondria in the encapsulated T cells was reduced, presumably due to the inhibition of antigen presentation leading to a reduced energy requirement of the T cells (online supplemental information, online supplemental figure S7C).

Encapsulated T cells mitigates GVHD in allogeneic transplantation AML model

On confirming the immune-isolation function of encapsulated T cells, it was crucial to explore whether encapsulation mitigates GVHD in AML mouse models post-transplantation. We established a mouse model for both AML and GVHD (figure 4A). Following transplantation of BMCs combined with encapsulated donor T cells, we observed noteworthy improvements in survival duration, GVHD score, and weight loss in AML recipient mice receiving encapsulated T-cell transplantation (Figure 4B–D).