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. 2023 Jun 2;9(22):eadg1082.
doi: 10.1126/sciadv.adg1082. Epub 2023 Jun 2.

Immune engineered extracellular vesicles to modulate T cell activation in the context of type 1 diabetes

Matthew W Becker  1 , Leeana D Peters  2   3 , Thinzar Myint  2   3 , Dylan Smurlick  1 , Andrece Powell  1 , Todd M Brusko  2   3   4 , Edward A Phelps  1   2   3
Affiliations

Immune engineered extracellular vesicles to modulate T cell activation in the context of type 1 diabetes

Matthew W Becker et al. Sci Adv. .

Abstract

Extracellular vesicles (EVs) can affect immune responses through antigen presentation and costimulation or coinhibition. We generated designer EVs to modulate T cells in the context of type 1 diabetes, a T cell-mediated autoimmune disease, by engineering a lymphoblast cell line, K562, to express HLA-A*02 (HLA-A2) alongside costimulatory CD80 and/or coinhibitory programmed death ligand 1 (PD-L1). EVs presenting HLA-A2 and CD80 activated CD8+ T cells in a dose, antigen, and HLA-specific manner. Adding PD-L1 to these EVs produced an immunoregulatory response, reducing CD8+ T cell activation and cytotoxicity in vitro. EVs alone could not stimulate T cells without antigen-presenting cells. EVs lacking CD80 were ineffective at modulating CD8+ T cell activation, suggesting that both peptide-HLA complex and costimulation are required for EV-mediated immune modulation. These results provide mechanistic insight into the rational design of EVs as a cell-free approach to immunotherapy that can be tailored to promote inflammatory or tolerogenic immune responses.

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Figures

Fig. 1.
Fig. 1.. EV isolation and characterization.
(A) Schematic overview of the workflow. Conditioned medium is processed through low-speed centrifugation and vacuum filtration to remove cells and cellular debris and then filtered through 300-kDa molecular weight cutoff (MWCO) UF devices to remove soluble proteins and concentrate the EV fraction. Concentrated medium is loaded onto a BE-SEC column using the ÄKTA Pure chromatography system. The first eluting fractions (EVs) are collected via an automated fraction collector and then subsequently concentrated using 100-kDa MWCO UF devices and used for further experiments. Contaminating proteins trapped by the BE-SEC column are eluted out during the column wash. (B) NTA showing representative particle concentration and size distribution of EVs derived from K562 A2/PPI cells. (C) Western blot analysis of EV isolates from K562 A2/PPI cells demonstrating the presence of small EV markers. n = 3. (D) Representative TEM image of K562 A2/PPI cell–derived EVs. Scale bar, 200 nm.
Fig. 2.
Fig. 2.. Validating an in vitro target:effector coculture model for EV engineering.
(A) Schematic representation of J-1E6 and K562 coculture. Yellow and gray circles represent endogenous peptides presented in HLA-A2 and exogenous PPI15–24 added into the coculture system, respectively. (B) IL-2 secretion from J-1E6 T cells showing activation from K562 A2/PPI cells, which is increased with exogenous peptide loading in HLA-A2. K562 cells lacking HLA-A2 fail to induce IL-2 secretion from J-1E6 T cells even in the presence of exogenous peptide. n = 3. Representative of four independent experiments. (C) Surface expression of the activation marker CD69 on J-1E6 T cells over a 24-hour coculture with K562 A2/PPI cells. n = 5. (D) Surface expression of PD-1 on J-1E6 T cells over a 24-hour coculture with K562 A2/PPI cells. n = 5. Statistical differences for (B) were determined by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Statistical differences for (C) and (D) were determined by two-way ANOVA followed by Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant. (A) was created using BioRender.com. MFI, median fluorescent intensity.
Fig. 3.
Fig. 3.. EVs need APCs to modulate T cell activation in vitro.
(A) Schematic representation of J-1E6 coculture with K562 A2/PPI cells and K-EVs. (B) IL-2 secretion from J-1E6 T cells showing increased activation with increasing amounts of K-EVs in cultures. Representative of five independent experiments. (C) IL-2 secretion from J-1E6 T cells after coculture with K562 A2/PPI cells and/or EVs, showing that EVs alone do not activate T cells. n = 3. (D) IL-2 secretion from J-1E6 cells after coculture with K562 A2/PPI cells or EVs, showing that EVs in the presence of exogenous PPI15–24 do not activate T cells. Representative of four independent experiments. (E) Schematic representations of the different conditions used in (F) to determine EV interactions in the coculture system. (F) Confocal microscopy images of DiR-labeled K-EVs incubated with either DiI-labeled J-1E6, DiO-labeled K562 A2/PPI, or both cell types for 24 hours. DiR signal was detected in cultures with K562 A2/PPI cells and J-1E6 with K562 A2/PPI cells but not J-1E6 cells alone, suggesting that K562 A2/PPI cells endocytose EVs. Representative of two independent experiments. (G) A high-resolution, three-dimensional micrograph of K562 A2/PPI cells incubated with DiR-labeled K-EVs. Yellow arrows indicate cell surface–bound EV clusters. (H) MFI quantification of confocal microscopy images, showing higher DiR EV signal in K562 A2/PPI cells and no significant difference between J-1E6 cells with or without K-EV coincubation. Each data point represents quantification from one image, with three images taken from each of two wells. Statistical differences for (B) through (D) and (H) were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, nonsignificant. (A) and (E) were created using BioRender.com. MFI, mean fluorescent intensity.
Fig. 4.
Fig. 4.. Driving PD-L1 expression in EV parent cells.
(A) Surface expression of PD-L1 on lentivirus-transduced cells shown via flow cytometry and further confirmed by Western blot analysis. (B) Schematic representation of J-1E6 coculture with either K562 A2/PPI or K562 PD-L1 cells, where PD-L1 engages with PD-1 to suppress IL-2 secretion. (C) IL-2 secretion from J-1E6 T cells showing decreased activation when cocultured with K562 PD-L1 cells compared to K562 A2/PPI. This suppression is still seen when exogenous PPI15–24 is added to cocultures. Representative of nine independent experiments. Statistical differences for (C) were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01. (B) was created using BioRender.com.
Fig. 5.
Fig. 5.. Validating PD-L1 expression in EVs.
(A) NTA showing representative particle concentration and size distribution of EVs derived from K562 PD-L1 cells. The inset shows the mode particle diameter for multiple K-EV and K–PD-L1–EV preparations, indicating that lentiviral transduction did not negatively affect EV generation. n = 13 and n = 12. (B) ELISA of PD-L1 on EVs from K562 cells compared to total EV protein content. n = 5. Data from two independent experiments. Statistical differences for (A) and (B) were determined by an unpaired, two-tailed t test. *P < 0.05. ns, nonsignificant.
Fig. 6.
Fig. 6.. K–PD-L1–EVs suppress T cell activation.
(A) Schematic representation of J-1E6 cocultures with K562 A2/PPI cells and K–PD-L1–EVs. (B) IL-2 secretion from J-1E6 cells showing decreased activation with K562 PD-L1 cells or increasing amounts of K–PD-L1–EVs in cultures, n = 3. Data shown are representative of three independent experiments. (C) Fold change in IL-2 secretion across all experiments with different EV treatments, showing that K-EVs significantly increase and K–PD-L1–EVs significantly decrease IL-2 secretion. T-EVs and T–PD-L1–EVs have no significant effect. Each dot represents a technical replicate from an independent experiment, and each color represents an independent experiment. Each technical replicate was normalized to its independent experimental control with just J-1E6 and K562 A2/PPI cells. Statistical differences for (B) were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical differences for (C) were determined using a linear mixed effects model where fold change in IL-2 is predicted by the EV treatment as a linear fixed effect and the individual experiment as a random effect. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, nonsignificant. (A) was created using BioRender.com.
Fig. 7.
Fig. 7.. EV PD-L1 reduces T cell–mediated target cell killing in vitro.
(A) Schematic representation of T-IGRP cocultures with K562 A2/PPI cells and K–PD-L1–EVs. (B) Specific lysis of K562 cells by T-IGRP avatars, showing a 60% reduction in specific lysis of K562 A2/PPI cells when K–PD-L1–EVs are added to cocultures, n = 3. Representative of two independent experiments. (C) Specific lysis of K562 A2/PPI cells by T-IGRP avatars, showing a 60 to 70% increase in specific lysis when K-EVs are added to cocultures. Representative of two independent experiments. Statistical differences for (B) were determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical differences for (C) were determined by unpaired, two-tailed t test. *P < 0.05, ***P < 0.001, ****P < 0.0001. (A) was created using BioRender.com.
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