
==== Front
J Immunother Cancer
J Immunother Cancer
jitc
jitc
Journal for Immunotherapy of Cancer
2051-1426
BMJ Publishing Group BMA House, Tavistock Square, London, WC1H 9JR

39242117
10.1136/jitc-2023-008663
jitc-2023-008663
Original Research
Clinical/Translational Cancer Immunotherapy
2435
1506
Immune isolation-enabled nanoencapsulation of donor T cells: a promising strategy for mitigating GVHD and treating AML in preclinical models
Mei Dan 10meidan225@outlook.com

Xue Ziyang 10935463041@qq.com

Zhang Tianjing 10ztianjing123@163.com

Yang Yining 10yangyining1999@163.com

Jin Lin 10jinlin@stu.ahmu.edu.cn

Yu Qianqian 11174407393@qq.com

Hong Jian 2hj911946@163.com

Zhang Xianzheng 13zxzhang0514@163.com

Ge Jinru 1972991073@qq.com

Xu Li 12586881295@qq.com

Wang Han 1488153922@qq.com

Zhang Ziwei 1884599603@qq.com

Zhao Yuchen 1384454353@qq.com

Zhai Yuanfang 11102624425@qq.com

Tao Qianshan 4ahmutqs@126.com

http://orcid.org/0000-0003-4060-7600
Zhai Zhimin 4zzzm889@163.com

Li Qingsheng 2wshsyh604@163.com

Li Hongxia 51262074653@qq.com

http://orcid.org/0000-0001-5070-8523
Zhang Lingling 1ll-zhang@hotmail.com

1 Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui, China
2 Department of Hematology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
3 Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
4 Department of Hematology, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
5 Department of Hematology and Oncology, The Third Affiliated Hospital of Anhui Medical University, Hefei, China
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

Supplement: Additional supplemental material is published online only. To view, please visit the journal online (https://doi.org/10.1136/jitc-2023-008663).

However, it should be noted that the technology discussed in this study is patented, with LZ and DM listed as inventors on the patent. All patent inventors are affiliated with the institutions listed in the author affiliations section. The authors affirm that this potential conflict of interest has not influenced the design, conduct, or reporting of the research presented in this manuscript. Full transparency is maintained regarding the authors’ involvement in the patented technology to ensure integrity and credibility in the publication process.

Professor; ll-zhang@hotmail.com
DM, ZX, TZ, YY and LJ contributed equally.

2024
05 9 2024
12 9 e00866313 8 2024
Copyright © Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
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Abstract

Background

In allogeneic-hematopoietic stem cell transplantation for acute myeloid leukemia (AML), donor T cells combat leukemia through the graft-versus-leukemia (GVL) effect, while they also pose a risk of triggering life-threatening graft-versus-host disease (GVHD) by interacting with recipient cells. The onset of GVHD hinges on the interplay between donor T cells and recipient antigen-presenting cells (APCs), sparking T-cell activation. However, effective methods to balance GVHD and GVL are lacking.

Methods

In our study, we crafted nanocapsules by layering polycationic aminated gelatin and polyanionic alginate onto the surface of T cells, examining potential alterations in their fundamental physiological functions. Subsequently, we established an AML mouse model and treated it with transplantation of bone marrow cells (BMCs) combined with encapsulated T cells to investigate the GVL and anti-GVHD effects of encapsulated T cells. In vitro co-culture was employed to probe the effects of encapsulation on immune synapses, co-stimulatory molecules, and tumor-killing pathways.

Results

Transplantation of BMCs combined with donor T cells selectively encapsulated onto AML mice significantly alleviates GVHD symptoms while preserving essential GVL effects. Encapsulated T cells exerted their immunomodulatory effects by impeding the formation of immune synapses with recipient APCs, thereby downregulating co-stimulatory signals such as CD28-CD80, ICOS-ICOSL, and CD40L-CD40. Recipient mice receiving encapsulated T-cell transplantation exhibited a marked increase in donor Ly-5.1-BMC cell numbers, accompanied by unaltered in vivo expression levels of perforin and granzyme B. While transient inhibition of donor T-cell cytotoxicity in the tumor microenvironment was observed in vitro following single-cell nanoencapsulation, subsequent restoration to normal antitumor activity ensued, attributed to selective permeability of encapsulated vesicle shells and material degradation. Moreover, the expression of apoptotic proteins and FAS-FAS ligand pathway at normal levels was still observed in leukemia tumor cells.

Conclusions

Encapsulated donor T cells effectively mitigate GVHD while preserving the GVL effect by minimizing co-stimulatory signaling with APCs through early immune isolation. Subsequent degradation of nanocapsules restores T-cell cytotoxic efficacy against AML cells, mediated by cytotoxic pathways. Using transplant-encapsulated T cells offers a promising strategy to suppress GVHD while preserving the GVL effect.

Graft versus host disease - GVHD
Graft versus leukemia
Nanoparticle
T cell
Anhui Provincial Department of Education Practice Program 2022cxcysj082 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China U1803129 The Third Affiliated Hospital of Anhui Medical University, Basic and Clinical Collaborative Research Enhancement Program Cultivation Special 2022sfy003 Anhui Key R&D Program 2021 - Population Health Project 202104j07020032 Anhui Medical University Postgraduate Research and Practice Innovation Program YJS20230009 Key Projects of Academic Funding for Top Talents in Disciplines (Majors) in Universities in Anhui Province gxbjZD2020060
==== Body
pmcWHAT IS ALREADY KNOWN ON THIS TOPIC

Previous research has highlighted the utility of cell encapsulation technology, which has been predominantly used in islet transplantation. However, while this technology has shown promise in various transplantation contexts, its application in preventing graft-versus-host disease (GVHD) remains an area that requires further exploration. Additionally, the importance of maintaining T-cell viability and functionality during transplantation to preserve the graft-versus-leukemia (GVL) effect has been well-established. Conventional transplantation methods often lead to complications such as GVHD due to interactions between donor T cells and recipient antigen presenting cells (APCs), necessitating innovative approaches to improve outcomes.

WHAT THIS STUDY ADDS

This study introduces a groundbreaking approach by effectively nano-encapsulating individual donor T cells using aminated gelatin overlaid with alginate. Highlighting the unique effect of single-cell nanoencapsulation on T cells, this technique preserves T-cell viability and essential cellular functions while providing immune isolation during the early stages of transplantation. The findings demonstrate that nano-encapsulated donor T cells mitigate the onset and progression of GVHD by reducing co-stimulatory signals with recipient APCs. Additionally, the study reveals that on the degradation of nanocapsule shells, the encapsulated T cells regain their cytotoxic efficacy against acute myeloid leukemia cells, thereby preserving the GVL effect.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

This study introduces several groundbreaking innovations with potential implications for research, practice, and policy. First, it sheds light on the unique effect of single-cell nanoencapsulation on T cells, offering a significant advancement in cell therapy techniques. Second, the development of nano-encapsulated T cells as a novel cell therapy product showcases a promising approach for enhancing the efficacy and safety of T-cell transplantation. Lastly, the establishment of a new transplantation model leveraging nano-encapsulated T cells provides a valuable tool for studying and potentially treating hematologic malignancies, informing future research directions.

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.811 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 methods

Reagents

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

Reagent or resource	Source	Identifier	
Antibodies	
 APC-conjugated anti-mouse LFA-1 (M17/4)	eBioscience	Cat#17-0111-82; RRID: AB_11217471	
 APC-conjugated anti-mouse CD154 (MR1)	eBioscience	Cat#17-1541-81; RRID: AB_795824	
 PE-conjugated anti-mouse CD80 (B7-1)	eBioscience	Cat#12-0801-81; RRID: AB_465751	
 PE-Cy7-conjugated anti-mouse H-2Kb (AF6-88.5)	BioLegend	Cat#116520; RRID: AB_2721684	
 AF647-conjugated anti-mouse H-2Kd (SF1-1.1)	BioLegend	Cat#116612; RRID: AB_493067	
 BV421-conjugated anti-mouse CD3 (17A2)	BioLegend	Cat#100228; RRID: AB_2562553	
 BV421-conjugated anti-mouse CD11c (N418)	BioLegend	Cat#117329; RRID: AB_10897814	
 FITC-conjugated anti-mouse I-A/I-E (M5/114.15.2)	BioLegend	Cat#107616; RRID: AB_493523	
 APC-conjugated anti-mouse CD54 (YN1/1.7.4)	BioLegend	Cat#116119; RRID: AB_10613645	
 PE-conjugated anti-mouse CD275 (HK5.3)	BioLegend	Cat#107405; RRID: AB_2248797	
 BV421-conjugated anti-mouse CD45.1 (A20)	BioLegend	Cat#110731; RRID: AB_10896425	
 BV510-conjugated anti-mouse CD45.2 (104)	BioLegend	Cat#109838; RRID: AB_2650900	
 PE-Cy5.5-conjugated anti-mouse CD25 (3C7)	BioLegend	Cat#101911; RRID: AB_10613642	
 AF647-conjugated anti-mouse Foxp3 (MF-14)	BioLegend	Cat#126407; RRID: AB_1089116	
 BV421-conjugated anti-mouse IL-17A (TC11-18H10.1)	BioLegend	Cat#506925; RRID: AB_10900442	
 APC-conjugated anti-mouse perforin (S16009B)	BioLegend	Cat#154403; RRID: AB_2721464	
 BV421-conjugated anti-mouse granzyme B (QA18A28)	BioLegend	Cat#396413; RRID: AB_2810602	
 LEAF Purified anti-mouse CD3 (145–2 C11)	BioLegend	Cat#100340; RRID: AB_11149115	
 LEAF Purified anti-mouse CD28 (145–2 C11)	BioLegend	Cat#102116; RRID: AB_11147170	
 FITC-conjugated anti-mouse CD4 (H129.19)	BD Biosciences	Cat#553650; RRID: AB_394971	
 PE-conjugated anti-mouse CD8 (53–6.7)	BD Biosciences	Cat#553032; RRID: AB_394571	
 APC-conjugated anti-mouse CD278 (C398.4A)	BD Biosciences	Cat#565883; RRID: AB_2744479	
 PE-conjugated anti-mouse CD40 (3/23)	BD Biosciences	Cat#561846; RRID: AB_395055	
 PE-conjugated anti-mouse CD3 (145–2 C11)	BD Biosciences	Cat#553063; RRID: AB_394597	
 PE-conjugated anti-mouse CD19 (1D3)	BD Biosciences	Cat#553786; RRID: AB_395050	
 PE-Cy5.5-conjugated anti-mouse lineage cocktail	BD Biosciences	Cat#561317; RRID: AB_10612020	
 Murine GM-CSF	PeproTech	Cat#315-03-20; RRID: AB_2085670	
 Murine IL-4	PeproTech	Cat#214-14-20; RRID: AB_317026	
 APC-conjugated anti-mouse CD28 (REA806)	Miltenyi	Cat#130-111-973; RRID: AB_2656964	
 FITC-conjugated anti-human CD68	BD Biosciences	Cat#562111; RRID: AB_10892822	
 PE-conjugated anti-human CD86	BD Biosciences	Cat#560957; RRID: AB_10563415	
 BV421-conjugated anti-human CD206	BD Biosciences	Cat#566281; RRID: AB_2739655	
 BB515-conjugated anti-human CD40	BD Biosciences	Cat#585927; RRID: AB_2802728	
 FITC-conjugated anti-human CD40L	BD Biosciences	Cat#566268; RRID: AB_2739646	
 FITC-conjugated anti-human CD28	BD Biosciences	Cat#535721; RRID: AB_2849621	
 PE-conjugated anti-human CD3	BD Biosciences	Cat#561803; RRID: AB_10893594	
 Beta actin antibody	Affinity Biosciences	Cat# AF7018; RRID: AB_2839420	
 Bcl2 rabbit pAb	Zen-Bio	Cat# 381702; RRID: AB_2924433	
 Bax rabbit pAb	Zen-Bio	Cat# 380709; RRID: AB_2924434	
 Rabbit anti-CD95/FAS antibody	Bioss	Cat# bs-6477R	
 Fas ligand rabbit pAb	Bioss	Cat# bs-0216R; RRID: AB_10856737	
 caspase-3 p17 subunit rabbit pAb	Bioss	Cat# bs-20364R	
 Goat anti-rabbit IgG (H+L)	Elabscience	Cat# E-AB-1003; RRID: AB_2921220	
Chemicals, peptides, and recombinant proteins	
 Gelatin	Sigma-Aldrich	Cat#G2625	
 Alginate	Sigma-Aldrich	Cat#A2158	
 5 (6)-isothiocyanate	Sigma-Aldrich	Cat#F3651	
 Busulfan	Sigma-Aldrich	Cat#B2635	
 Cyclophosphamide	Sigma-Aldrich	Cat#PHR1404	
 FITC-dextran	Sigma-Aldrich	Cat#BCCC2378	
 Hexamethyldisilane	Macklin	Cat#H810965	
 2-(N-morpholino) ethanesulfonic	Macklin	Cat#M813439	
 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide	Macklin	Cat#N808856	
 N-hydroxysulfosuccinimide	Solarbio	N/A	
 Z-IETD-FMK	MCE	CAS#210344-98-2	
Critical commercial assays	
 Mice ELISA Assay Kits	Fcmacs Biotech	Cat#FMS-ELM028, FMS-ELM003, MS-ELM027	
 Mice ELISA Assay Kits	MULTISCIENCES	Cat#EK268, EK206, EK210, EK280	
 FITC annexin V Apoptosis Detection Kit I	BD Biosciences	Cat#556 547	
 Cell Counting Kit-8	Beyotime	Cat#C0037	
 Anti-PE MicroBeads	Miltenyi	Cat#130-048-801	
 CFSE Cell Proliferation Assay Kit	eBioscience	Cat#65-0850-84	
Lentiviral	
 LV16 (U6/Luciferase17&Puro)	GenePharma	Cat# D02001	
Experimental models: cell lines	
 WEHI-3B	SHRBIO Biological	RRID: CVCL_2239	
 C1498	SUNNCELLBiological	RRID: CVCL_3494	
Experimental models: organisms/strains	
 C57BL/6	Beijing Sipeifu Biotechnology	Cat# B204	
 BALB/c	Beijing Sipeifu Biotechnology	Cat# B201	
 C57BL/6 CD45.1	Nanjing Junke Bioengineering	Cat# JKM0451	
Software and algorithms	
 GraphPad Prism	GraphPad Software	RRID:SCR_002798	
 FlowJo	FlowJo	RRID:SCR_008520	
 ImageJ	NIH, USA	RRID:SCR_003070	
APCantigen-presenting cell CFSEcarboxyfluorescein succinimidyl esterFITCfluorescein isothiocyanate

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 tables36.

Table 2 Assessment of clinical graft-versus-host disease in transplanted animals

Criteria	Grade 0	Grade 1	Grade 2	
Weight loss	<10%	>10% to <25%	>25%	
Posture	Normal	Hunching noted only at rest	Severe hunching impairs movement	
Activity	Normal	Mild-to-moderately decreased	Stationary unless stimulated	
Fur texture	Normal	Mild-to-moderate ruffling	Severe ruffling/poor grooming	
Diarrhea	No	Mild-to-moderate diarrhea	Severe diarrhea	
Skin integrity	Normal	Scaling of paws/tail	Obvious areas of denuded skin	

Table 6 Assessment of H&E-stained images of the colon from transplanted animals

	Grade	Standard description	
Degree of mucosal injury	0	Normal colonic mucosa	
1	One-third of the crypt glands were lost	
2	Two-third of the crypt glands were lost	
3	All the crypt glands were lost	
4	The mucosal epithelium was eroded and destroyed, accompanied by obvious inflammatory cell infiltration	
Extent of mucosal injury	0	Normal colonic mucosa	
1	Local mucosal damage	
2	Damage involved one-third of the colon	
3	Damage involved two-third of the colon	
4	Damage involved all the colon	

Table 5 Assessment of H&E-stained images of skin from transplanted animals

Grade	Standard description	
0	No hyperkeratosis or insufficiency was found in the stratum corneum, and no small abscess of Munro was found.	
0.5	Hyperkeratosis was found in the stratum corneum, slight prolongation of the dermal process, and slight infiltration of single or multinucleate cells in the dermis.	
1	Keratosis was found in the stratum corneum, the granular layer in the epidermis was thinner or disappeared, the acanthoid layer was thickened, the dermal process was moderately prolonged, and the single or multinucleate cells were moderately infiltrated in the undulating dermis.	
2	Munro abscess was found in the stratum corneum with a moderately prolonged dermal process and moderate infiltration of mononuclear or multinucleate cells in the undulating dermis.	
3 or higher	Munro small abscess was found in the stratum corneum, the dermal process was severely prolonged, the undulating dermis was heavily infiltrated by single or multinucleate cells. If the superior roof of the mastoid is found, add 0.5, if telangiectasia is found, add 0.5.	

Table 4 Assessment of H&E-stained images of spleen from transplanted animals

Grade	Standard description	
0	The boundary between red pulp and white pulp was clear, the germinal center and marginal zone were clear, and there was no abnormality, hyperplasia, atrophy, or hyalinization.	
1	Splenic corpuscles were visible, germinal center, red pulp and white pulp were clearly demarcated, no obvious congestion was observed, and inflammatory cell infiltration was reduced.	
2	The splenosomes of the spleen were blurred, the boundary between the germinal center, red pulp and white pulp was still clear, and some congestion and inflammatory cell infiltration were seen.	
3	The structure of the white and red pulp was disordered, without clear boundaries and could not be identified. The splenic sinus was dilated, the interstitial hemorrhage was severe, there was obvious congestion, and the number of lymphocytes was significantly reduced.	

Table 3 Assessment of H&E-stained images of liver from transplanted animals

Criteria	Grade 0	Grade 1	Grade 2	Grade 3	Grade 4	
Portal inflammation	None	Minimal	Mild	Moderate	Severe	
Lobular inflammation	None	Minimal	Mild	Moderate	Severe	
Fibrosis	None	Portal	Periportal	Bridging fibrosis	Cirrhosis	

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.

Results

Functional 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.1618 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).

Figure 4 Transplantation of encapsulated T cells in combination with BMCs inhibits the development of GVHD in recipient mice while preserving the GVL effect. (A) Experimental design diagram, demonstrating the use of T cells encapsulated with BMCs for the inhibition of GVHD progression in female BALB/c recipient mice. Mice (20 g each) were administered intraperitoneal (IP) injections of 0.4 mg busulfan and 2 mg cyclophosphamide 7 days prior to BMT. WEHI-3B cells were subsequently infused via tail vein 1-day pre-transplantation. The transplantation involved injecting BMCs (CD45.1) and splenic T cells (CD45.2) from H2-b C57BL/6 mice into the recipient's tail veins. (B–D) Graphical representation of alterations in body weight, clinical scores, and survival rates of mice across all groups, monitored over a period of 60 days. (E–H) Flow cytometry scatter plots and associated statistical results, indicating the proportions of CD3+, CD4+, and CD8+T cell subsets in mice peripheral blood. A comparison is made between groups receiving encapsulated and non-encapsulated T cells. (I) Flow cytometry quantification of cells derived from CD45. One donor mice in recipient mice peripheral blood reflects donor bone marrow-derived cell engraftment. Splenic lymphocytes from recipient mice were analyzed. (J–K) Representative scatter plots depicting Treg and T helper cell 17 cell subsets. (L) Representative flow cytometry peak plots and statistical analysis of CD28, CD40L, and ICOS expression within the H2kb+CD3+ subset. (M) Representative flow cytometry peak plots and statistical analysis of CD80, CD40, and ICOSL expression within the H2kd+LIN-CD11c+MHC-II+ subpopulation was presented. (N–Q) Bar graphs representing the secretion levels of IL-6, IL-10, IFN-γ, and C-X-C motif chemokine ligand 10 in plasma and peritoneal macrophages across both encapsulated and non-encapsulated groups. (S–V) Statistical plot of pathological scores of H&E-stained images of each target organs from mice. Pooled data from three independent experiments each with seven recipients. Survival (D) Kaplan-Meier curve, clinical score (C) weight (B) from two or three independent experiments, each with seven mice per group, are shown. Mean value±SEM; *p<0.05, ***p<0.001. BMC, bone marrow cell; GVHD, graft-versus-host disease; IFN, interferon; IL, interleukin; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; Treg, regulatory T cell.

In the peripheral blood of the encapsulated group, we detected higher numbers and proportions of CD3+T cells compared with the non-encapsulated group, with a greater proportion of CD3+CD8+ T cells and a lower proportion of CD3+CD4+ T cells than the non-encapsulated group (figure 4E–H). Intriguingly, we discovered a higher number of BMCs from C57BL/6-Ly-5.1 donor mice in the encapsulated group than in the non-encapsulated group (figure 4I). On day 21, we evaluated the recipient mice for peripheral blood hematological expression. The proportion of lymphocytes and the count of leukocytes and platelets in the peripheral blood of the encapsulated group were significantly higher and closer to the normal control group, whereas the proportion of eosinophils and neutrophils was significantly lower (online supplemental information, online supplemental figure S8). These findings suggest that our transplantation of encapsulated T cells may facilitate improved hematopoiesis in recipient mice.

We also analyzed the impact of nanoencapsulation on the balance of Th17 and Tregs in recipient mice. We found a higher CD4+CD25+FoxP3+ ratio in the encapsulated T-cell group than in the non-encapsulated group. Correspondingly, the proportion of CD4+IL-17+ in the encapsulated group was lower than in the non-encapsulated group (figure 4J,K), with the difference between the two groups being statistically significant.

In our in vitro studies, we demonstrated that nanoencapsulation inhibits the interaction between donor T cells and recipient APCs. To further validate this finding in vivo, we analyzed spleens derived from donor-derived T cells and recipient-derived DCs to detect the expression of co-stimulatory molecules (online supplemental information, online supplemental figure S9). Expression of the surface co-stimulatory molecules CD28, ICOS, and CD40L was significantly lower in splenic T cells from the encapsulated T-cell group compared with the non-encapsulated group (figure 4L). Similarly, the expression of surface co-stimulatory molecules CD80, ICOSL, and CD40 in splenic DCs of encapsulated T-cell mice was significantly lower than in the non-encapsulated group (figure 4M).

The release of inflammatory cytokines plays a pivotal role in the progression of GVHD. We collected recipient peripheral blood serum and peritoneal macrophages and found that the secretion of IFN-γ, IL-6, and IL-10 was significantly reduced in the encapsulated T-cell group. Moreover, the expression of the CXCL10 was lower than in the non-encapsulated group (figure 4N–Q).

Pathological analysis of tissue samples revealed that the target organs of the recipient mice in the non-encapsulated group were inundated with inflammatory cells. Fibrous septa formed between hepatic blood vessels, and hepatic sinusoids showed dilatation and bruising. Epithelial cells of the colonic mucosa appeared disorganized, some ruptured and damaged or even detached, with the crypt structure having disappeared. There was hyperkeratosis of the skin stratum corneum, thickening of the granular layer and stratum spinosum, and excessive prolongation of the spinous processes (figure 4R–V). However, these pathological symptoms were less severe in the encapsulated T-cell group, corroborating the above clinical score data (figure 4C).

Single-cell encapsulation preserves the cytotoxic effects of T cells against leukemia tumor cells

The WEHI-3B AML cell model facilitated a comparative analysis of GVL effects between encapsulated and non-encapsulated T cells. Remarkably, the BMCs group, which was transplanted with only BMCs, exhibited significant leukemia tumor growth by day 27. In contrast, neither the encapsulated nor non-encapsulated T-cell groups showed any detected proliferation or metastasis of tumor cells throughout the 60-day observation period (figure 5A,B) perforin and granzyme B expressions in T cells from peripheral blood were scrutinized. The results revealed no notable difference in the expression ratio of these elements between the encapsulated and non-encapsulated groups, regardless of whether the cells were CD4+or CD8+ T cells. Consequently, we postulated that encapsulated donor T cells might treat AML mice via the perforin-granzyme pathway (figure 5C–E).

Figure 5 The cytotoxic effect of encapsulated T cells on WEHI-3B cells differed temporally from that of non-encapsulated T cells. WEHI-3B-Luc cells were injected and transplanted into three groups: BMCs, encap, and non-encap for treatment, with GVL effects assessed via bioluminescence imaging. (A) Representative fluorescence signal images of WEHI-3B-Luc in each group post-administration of luciferin substrate on day 27 (D27) are presented. (B) Quantitative analysis was conducted using the average emissivity of the animals (p/s/cm²/sr). (C–D) Representative flow cytometry plots depict perforin and granzyme B expression in CD4+and CD8+ T cells in the peripheral blood of recipient mice. (E) Scatter plot illustrating the ratio of perforin and granzyme B expression in CD4+ and CD8+ T cells between encapsulated and non-encapsulated groups. CFSE-labeled tumor cells were co-cultured with T cells at a 1:10 ratio. The impact of encapsulated and non-encapsulated T cells on (F, H) WEHI-3B cells and (G, I) C1498 cells proliferation was assessed at 24 hours and 72 hours. The attenuation of cell proliferation fluorescence was observed relative to the fluorescence at 0 hours. The effects of encapsulated and non-encapsulated groups on WEHI-3B cell apoptosis were examined using membrane-bound protein-PI staining at 24 hours, 48 hours and 96 hours. (J) Representative flow cytometry plots of WEHI-3B cells from both groups at 24 hours, 48 hours, and 96 hours are depicted. (K) Statistical analysis comparing the proportion of early apoptotic+late apoptotic WEHI-3B cells is presented. Mean value±SEM, results of at least five repeat experiments each with three samples or eight recipients. *p<0.05, ***p<0.001, NS, no significant difference. BMC, bone marrow cell; CFSE, carboxyfluorescein succinimidyl ester; FITC, fluorescein isothiocyanate; GVL, graft-versus-leukemia.

To investigate the influence of nanoencapsulation on leukemia tumor cell apoptosis, we co-cultured T cells with WEHI-3B cells or C1498 cells. Initial findings indicated that both at 24 hours and 48 hours, the encapsulated T-cell group had a significantly lower rate of leukemia tumor cell apoptosis compared with the non-encapsulated group. However, by the 96-hour mark, the apoptosis rate of leukemia tumor cells did not significantly differ between the two groups. The trends observed in these results were corroborated in two types of leukemia tumor cells, namely WEHI-3B and C1498 (figure 5J,K and online supplemental information, online supplemental figure S10A,B). Moreover, this temporal variation in the tumor-killing effect of the encapsulated T cells was further validated by the CFSE proliferation assay (figure 5F–I).

Encapsulated T cells retain the GVL effect through nanomembrane degradation retain GVL effect through nanomembrane degradation

The encapsulated T cells were not individually dispersed, implying that the encapsulation may have preserved cell-to-cell information exchange. We hypothesized that temporal variations in the immune isolation and tumor-killing effects of encapsulated T cells might be linked to the degradation of the encapsulation material.

To explore the impact of encapsulated T cells on WEHI-3B cell viability, we implemented co-culturing of WEHI-3B cells with T cells and also employed Transwell (0.4 µm) to separate the two types of cells. In the direct co-culture, WEHI-3B cell viability was initially higher in the encapsulated T-cell group compared with the non-encapsulated group from 24 hours to 48 hours, but by 72 hours the levels were comparable (figure 6A). Intriguingly, in the Transwell co-culture, there was no significant difference in WEHI-3B cell viability from 24 hours to 72 hours between the two groups (figure 6B). The aforementioned results were replicated in the Transwell co-culture experiment involving C1498 cells and encapsulated T cells (online supplemental information, online supplemental figure S10C). Furthermore, consistent trends and outcomes were noted in the high-content imaging experiments conducted on WEHI-3B cells (online supplemental information, online supplemental figure S10D,E). Subsequently, we examined the expression of perforin and granzyme B in CD4+T cells and CD8+T cells in in WEHI-3B co-culture system and C1498 co-culture system. After 24 hours, encapsulated T cells showed a decline in both perforin and granzyme expressions compared with the non-encapsulated T cells, although the difference was not statistically significant. By the 72-hour mark, the encapsulated T-cell group indicated increased granzyme expression, still slightly lower than the non-encapsulated group, with the difference again not statistically significant. However, perforin expression in the encapsulated T-cell group was significantly lower than in the non-encapsulated group (figure 6C–H). Although the reduction in perforin expression by CD8+T cells following 72 hours in the C1498 co-culture system did not exhibit significant variance from that of non-encapsulated T cells, overall findings were largely consistent with the above description (online supplemental information, online supplemental figure S11).

Figure 6 Degradable encapsulated vesicle shells are selectively permeable to macromolecules in T cells. Cell viability of WEHI-3B cells co-cultured with T cells assessed using CCK-8 at various time points over 72 hours, either (A) directly or (B) separated by a Transwell (0.4 µm) barrier at a ratio of 1:10. (C–D) Representative flow cytometry images depicting perforin expression on CD4+ and CD8+ T cells at 24 hours and 72 hours, respectively. (E–F) Representative flow cytometry images demonstrating granzyme B expression on CD4+ and CD8+ T cells at 24 hours and 72 hours, respectively. (G–H) Expression levels of perforin and granzyme B in T-cell subsets at different time points in the co-culture system. (I) Single-cell encapsulation of T cells using FITC-alginate, seeded into a 24-well culture plate at 2×105 cells/mL, and monitoring of T-cell extravasation at 0, 24, 48, and 96 hours by FITC fluorescence changes in encapsulation materials. Assessment of permeability in encapsulated T cells: (J) FITC-dextran (40 kD) incubation with 2-layer and 4-layer encapsulated T cells for 24 hours, measured by flow cytometry to determine diffusion, and (K) incubation of reactive oxygen species fluorescent probe (DCFH-DA) with 2-layer and 4-layer encapsulated T cells for 24 hours, with average fluorescence intensity measured by flow cytometry to evaluate encapsulating effects on T-cell permeability. Mean value±SEM, results of at least five repeat experiments each with three samples. **p<0.01, ***p<0.001, NS, no significant difference. FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; ROS, reactive oxygen species.

To investigate the correlation between T-cell proliferation and encapsulation material degradation, we initially used FITC-labeled alginate for T-cell encapsulation. Following encapsulation with FITC-alginate, degradation of the fluorescent outermost encapsulation material commenced at the 48-hour mark and was nearly complete by 96 hours (figure 6I). Interestingly, the encapsulated T cells remained closely aggregated, suggesting that the encapsulation might facilitate cell-to-cell communication. Additionally, we employed CFSE and Cy3-labeled alginate simultaneously to monitor material degradation during T-cell proliferation. It was evident that encapsulated T cells expanded, leading to the dispersion of alginate spacers on their surfaces (online supplemental information, online supplemental figure S12). To further elucidate the mechanism by which encapsulated T cells maintain the GVL effect, western blot analysis following 48 hours of in vitro co-culture revealed the presence of Bcl2, Bax, and cleaved-caspase-3 apoptotic proteins in WEHI-3B cells. Moreover, the FAS-FAS ligand (FASL) pathway exhibited no significant differences between the encapsulated and non-encapsulated T-cell groups (online supplemental information, online supplemental figure S13A,B,E,F). Similar trends were observed in C1498 cells (online supplemental information, online supplemental figure S13C,D,G,H). Furthermore, when encapsulated or non-encapsulated T cells were pre-treated with granzyme B inhibitors and subsequently co-cultured with WEHI-3B cells, expression of apoptotic proteins and the FAS-FASL pathway remained detectable in WEHI-3B cells (online supplemental information, online supplemental figure S13I–L). These findings indicate that encapsulated T cells largely do not influence the expression of apoptosis-related proteins in leukemia tumor cells, and the FAS-FASL pathway represents one of the mechanisms by which encapsulated T cells retain the GVL effect.

Given that perforin has a molecular weight between 70–75 kD and granzyme around 30 kD, we speculated that the porous membrane structure of the outer layer of encapsulated T cells might selectively permit molecules of different sizes. To test the permeability of differently layered encapsulated T cells, we used FITC-dextran with a molecular weight of 40 kD and incubated it with T cells encapsulated in layers 2 and 4 for 24 hours under light protection, using non-encapsulated T cells (Control-F) as a control. The rate of dextran diffusion was found to be 79.2% for 2-layer-encapsulated T cells, and 60.1% for 4-layer-encapsulated T cells (figure 6J). Notably, there was no significant difference in the reactive oxygen species diffusion assay between the two groups of cells (figure 6K). These findings highlighted that the porous membrane structure of encapsulated T cells modulates the diffusive transport of macromolecules in T cells.

Discussion

As it stands, the prevention and specific treatment of GVHD still leaves a lot to be desired. A variety of immunosuppressive drugs such as corticosteroids, calcium-modulated phosphatase inhibitors, methotrexate, and cytokine antagonists have shown promise but often leave the patient susceptible to a recipient of opportunistic infections, either fungal, bacterial, or viral.1921 T-cell removal from the transplant inoculum almost fully halts GVHD’s development, yet it brings along an increased likelihood of graft rejection and disease recurrence.22 The potential of antigen-activated donor T-cell inhibition to alleviate GVHD severity is well-documented, leading to the development of specialized T cells for transplantation therapy.23 24 These include invariant natural killer T cells, inducible Tregs, and engineered T cells. Despite encouraging results in the treatment of GVHD, the widespread use of these cells is still hampered by the high cost of their preparation.25 26 Given these limitations, our research has pivoted towards developing more cost-effective and feasible cell therapy techniques, with our focus primarily on encapsulating donor T cells in nanoscale selectively permeable microcapsules to isolate their interaction with APCs.

Cell encapsulation technology emerged in the 1980s as a novel method of safeguarding allografts of pancreatic islet cells from immune rejection through encapsulation within micrometer-sized hydrogel beads.27 28 Advances in this technique have led to single-cell nanoencapsulation of mammalian cells, following successful encapsulation of platelets.2933 For encapsulation, we opted for naturally degradable gelatin and alginate due to their biocompatibility. Gelatin, in particular, is useful for mimicking the extracellular matrix, providing an ideal environment for the encapsulated cells. The LbL single-cell encapsulation technique provides an artificial microenvironment for individual T cells, without limiting cellular functions to cell colonization. In our study, T cells encapsulated by four layers of LbL demonstrated a zeta potential similar to that of the non-encapsulated cells. Morphologically, the surface of T cells was smoother and appeared as a nanoscale encapsulated encapsulating with a porous structure in which microvilli on the T cells were embedded, and this structure provided for the exchange of oxygen, nutrients, and metabolites by the T cells. Furthermore, the procedure of encapsulation maintained its benign nature, evidencing no noteworthy influence on various vital aspects of T-cell functionality. These included, but were not limited to, the preservation of T-cell viability, the unaffected rhythm of proliferation, the retention of antibody binding efficacy, and the consistent secretion levels of integral cytokines such as TNF-α, IL-2, and IFN-γ.

IL-10, an immune regulatory factor produced by various cell types, holds the potential to predict the development of GVHD. However, it is not necessarily indispensable for alleviating GVHD.34 35 Elevated serum IL-10 levels have been associated with fatal outcomes in patients post-bone marrow transplantation, although the immunomodulatory role of IL-10 in GVHD remains ambiguous.36 In our study, we observed differences in IL-10 data compared with the general expectations based on previous research. Given that IL-10 levels significantly increase from the post-transplantation aplastic phase to the white blood cell recovery phase,37 we speculate that the mice in the non-encap T group may have been in this stage, while the mice in the encap group could survive for 60 days or longer. Furthermore, in the GVHD model, the initial inhibitory role of IL-10 may be exerted through APC-mediated strong immunosuppression on initial T cells, where its immunostimulatory effect predominates.38 From the perspective of encapsulation, the immunostimulatory effect of encapsulated T cells on APCs may be inhibited, affecting IL-10 secretion. Therefore, it is conceivable that the differences in IL-10 levels between the two groups of recipient mice manifest at specific stages of GVHD progression and may fluctuate over time.

T-cell activation involves the T cell receptor (TCR) initially binding to an antigen, following which various co-stimulatory and co-inhibitory signals mediated by T cell and APC receptors become active.39 40 Research in HSCT and related hematological fields has focused on the influence of these co-stimulatory signals on T-cell activation and GVHD.4144 For instance, gastrointestinal GVHD patients were found to have an increased count of ICOSL+plasma cell-like DCs associated with CD146+CCR5+ T cells.45 Additionally, the co-stimulatory molecule inhibitor abatacept, which blocks CD28, leading to signal inhibition, has heralded significant advancements in GVHD therapeutic studies.46 In our laboratory-based experiments, nanoencapsulation successfully inhibited co-stimulatory signal expression on T cells without affecting the co-stimulatory molecules present on DC surfaces. However, in live studies on the encapsulated group of recipient mice, we observed reduced expression of surface co-stimulatory molecules on spleen T cells in conjunction with DCs, including CD28-CD80, ICOS-ICOSL, and CD40L-CD40. The discrepancy between the in vitro and in vivo findings might be ascribed to the encapsulating envelope to modulate the in vivo inflammatory environment following chemotherapy. This could subsequently curb the maturation and activation of DCs within the recipient mice. Transitioning to the mobility of T cells, it is worth noting that the LFA-1 molecule is essential for T cells to translocate to the target organ and has a significant influence on the formation of immune synapses between T cells and APCs. In a similar vein, encapsulated T cell was found to curtail the expression of LFA-1 on splenic T cells and MHC-II and ICAM-1 on DCs in recipient mice. In addition, we expanded on our prior work by collecting humanized specimens for in vitro mixed lymphocyte reactions. Our findings indicate that the expression levels of LFA-1 and CD28 on encapsulated CD3+T cells, along with CD86+ on CD68+ macrophages, were all notably lower compared with non-encapsulated T cells, despite statistically significant differences observed in CD40 and CD40L expression (online supplemental information, online supplemental figure S14). These results underscore the potential of encapsulated T cells in providing immune isolation within in vitro modeling studies of human samples.

In HSCT, Tregs play a crucial role in modulating allogeneic responses, presenting potential strategies to prevent both acute and chronic GVHD.47 48 It has been discovered that encapsulated donor T cells can effectively manage the ratio of CD4/CD8 T-cell subsets and the Th17/Treg cell ratio within the CD4+Th cell population in recipient mice. This modulation promotes the differentiation of T cells towards CD4+Th and Tregs, thereby potentially mitigating GVHD. Consequently, the induction of Tregs subpopulation differentiation in the presence of co-stimulatory blockade may be an indirect effect resulting from inflammation suppression. In addition to these findings, the study observed a significant increase in cell count derived from C57BL/6-Ly-5.1 donor BMCs in the recipient mice of the encapsulated T-cell transplantation group. This observation suggests that encapsulated T cell may promote the restoration of the recipient’s hematopoietic function. Moreover, following the same encapsulation procedure, we opted to co-encapsulate donor T cells with BMCs for transplantation therapy. Interestingly, we discovered that encapsulated donor BMCs did not induce substantial changes in the recipient’s peripheral blood hematology (online supplemental figure S8). We co-cultured the co-encapsulated group with WEHI-3B cells and assessed apoptosis and cell viability. The results were comparable to those observed with encapsulated T cells, indicating that co-encapsulation with BMCs preserved the GVL effect of T cells (online supplemental figure S15A–C). This observation bears significant implications for future encapsulated bone marrow HSCT therapies, as it could circumvent the high costs associated with T-cell isolation during HSCT.22 49

The interaction between effector and target cells prompts the cytosolization in CTL cells of particles encapsulating potential cytolytic effector molecules. Perforin and granzyme B stand out as the most notable components of these particles.50 Studies have shown that perforin-deficient mice exhibit a higher susceptibility to spontaneous lymphoma.51 In the present study, it was revealed that the porous structure on the surface of encapsulated T-cell impeded the short-term release of the larger molecular weight perforin (MW=70–75 kD) but did not impact the release of the smaller molecular weight granzyme B (MW=30 kD). Moving to peripheral blood examination, no considerable variations were found in the expression of either perforin or granzyme B on CD4+T cells or CD8+T cells in mice receiving encapsulated donor T grafts compared with the non-encapsulated group. Consequently, these findings lead us to infer that single-cell nanoencapsulation, despite transiently inhibiting the cytotoxic effects of donor T cells in the tumor environment, did not permanently impede these effects. As the encapsulating capsules dissociating, the antitumor activity of encapsulated T-cell gradually returns to baseline levels. Furthermore, even with single-cell encapsulation of T cells, expression of apoptotic proteins and the FAS-FASL pathway was still observed in leukemia tumor cells. These results demonstrate that encapsulated T cells minimally affect the expression of leukemia tumor cell apoptotic proteins and can exert the GVL effect through various pathways including perforin, granzyme, and FAS-FASL. Acute GVHD (aGVHD) generally occurs during the initial stages post-receipt of allografts.52 Interestingly, encapsulated T-cell demonstrated a remarkable capability to evade allogeneic rejection during this critical period. We postulate that, once the encapsulating shell has dissociated, CTL surface antigens become exposed and selectively destroy tumor cells. Meanwhile, the period before the complete dissociation of the encapsulated vesicle provides a so-called “golden period” for the endothelial cells damaged by GVHD to undergo repairs, thereby re-establishing barrier function. By strategically exploiting this temporal discrepancy, the encapsulated donor T cells were able to mitigate the progression of GVHD while preserving the GVL effect of T cells during HSCT.

Conclusion

In summary, our experiments successfully led to the single-cell nanoencapsulation of T cells, safeguarding both their viability and fundamental cellular operations. The creation of an immune isolating barrier by the encapsulated T-cell minimized the expression of co-stimulatory molecules between donor T cells and recipient APCs, thus influencing the establishment of immune synapses. Furthermore, the encapsulation layer facilitates the passage of small molecules and specific cytokines without causing prolonged inhibition. Consequently, it enables the exertion of GVL effects through various pathways, including perforin, granzyme, and FAS-FASL. During HSCT, encapsulated T-cell strategically leveraged the temporal gap associated with the degradation of encapsulated capsids, which ultimately led to the suppression of aGVHD progression while preserving the GVL effect. This enlightening discovery paves the way for a robust theoretical foundation and offers a novel strategy for the employment of immune isolation technology and immune cells. This strategy could potentially revolutionize the treatment and prevention of aGVHD and the targeted extermination of tumor cells.

Highlights

This study introduces novel techniques, cellular therapeutic products, and migration models. The emphasis primarily lies on the research findings as follows:

Nanoencapsulation maintains T-cell functional integrity and viability.

Donor T-cell encapsulation mitigates graft-versus-host disease in acute myeloid leukemia mice, sustaining graft-versus-leukemia.

Nanoencapsulation curbs co-stimulatory molecule expression, impacting immune synapse formation.

Selective permeability of encapsulated T cells exerts cytotoxic effects on leukemia cells through various pathways, including perforin, granzyme, and FAS-FAS ligand.

supplementary material

10.1136/jitc-2023-008663 online supplemental file 1

Acknowledgements

We extend our sincere thanks to the Research Center and Electron Microscopy Center of Anhui Medical University for their technical support with SEM and TEM. We also appreciate the support of Professor Li Qingsheng and Dr Hong Jian from the Blood Center at the First Affiliated Hospital of Anhui Medical University for providing human peripheral blood samples.

Data availability statement

Data are available upon reasonable request.

Funding: This study was supported by the National Natural Science Foundation of China (Grant number U1803129), Anhui Key R&D Program 2021-Population Health Project (202104j07020032), The Third Affiliated Hospital of Anhui Medical University, Basic and Clinical Collaborative Research Enhancement Program Cultivation Special (2022sfy003), Anhui Provincial Department of Education Practice Program (2022cxcysj082), Key Projects of Academic Funding for Top Talents in Disciplines (Majors) in Universities in Anhui Province (gxbjZD2020060) and Anhui Medical University Postgraduate Research and Practice Innovation Program (YJS20230009).

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: All clinical research protocols were approved by the Research Ethics Committee of Anhui Medical University (No. 20180011). Participants gave informed consent to participate in the study before taking part.
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