OMVs from E. coli MG1655 Δpal ΔlpxM were isolated as previously described (32), with some modifications. Ultracentrifugation was used to isolate OMVs via pelleting at 235,000 x g for 2 hours at 8°C using a fixed angle 45 Ti rotor (Beckman Coulter). Pellets were washed via resuspension in PBS and pelleting again, before final resuspension in PBS.

The size and concentration of OMVs was measured by nanoparticle tracking using a NanoSight NS300 (Malvern Panalytical) and analysed by NanoSight NTA software (Malvern Panalytical). Where possible samples were diluted in PBS to obtain between 20 and 80 particles per frame. Results consisted of five measurements each using 60 second recordings. Camera sensitivity, gain and detection threshold were set to 16, 10 and 4 respectively, whilst samples were administered and recorded under controlled flow using the NanoSight syringe pump and script control system.

Whole blood from six healthy donors (HD1-HD6) stored in sodium citrate was purchased from Cambridge Bioscience and delivered<24 hours after sampling. Upon receipt, PBMCs were immediately isolated via ficol density gradient separation. Samples were then washed in supplemented RPMI 1640 media and PBS, before being resuspended in media for counting (NC3000 nucleocounter, Chemometec). All supplemented RPMI 1640 (Life Technologies) contained, 10% heat-inactivated foetal bovine serum (FBS) (One Shot, Gibco) and 2 mM L-glutamine (Gibco).

PBMCs were cultured in supplemented pre-warmed RPMI 1640 media at a density of 1x10⁶/ml. Cells were dosed with 100 μl of E. coli MG1655 Δpal ΔlpxM OMVs at a concentration of 1x10⁹/ml or 1x10¹⁰/ml to give a ratio of 1x10³:1 or 1x10⁴:1 (OMVs : PBMCs) respectively. Samples were incubated for either 24 hours or 5 days at 37°C and 5% CO₂, after which cells were pelleted for cell surface marker analysis by flow cytometry, and the supernatant used for cytokine analysis.

Quantification of cytokine production was determined by ELISA. IFN-γ analysis used an IFN gamma Human Uncoated ELISA Kit (ThermoFisher Scientific). Granzyme B was measured via an Ella Automated Immunoassay System (Ella, Protein Simple) using a 16 x 4 Custom Simple Plex Assay Panel, performed as per the manufacturer’s instructions. A custom ELISA array kit (Multi-Analyte ELISArray, Qiagen) was also used to screen a panel of cytokines and chemokines. For the ELISArray, cytokine concentration was measured via absorbance at OD_450nm normalised to a positive control sample as directed by the protocol provided.

1ml of PBMCs (1x10⁶/ml) from three healthy donors were incubated for 10 days with either 2x10¹⁰ E. coli MG1655 Δpal ΔlpxM OMVs (20000:1 ratio), 5 μM zoledronate (zoledronic acid monohydrate, Sigma-Aldrich) or PBS. In all cases, cells were also supplemented with 100 IU/ml IL-2 (human IL-2 IS premium grade, Miltenyi Biotec), which continued every three days along with re-adjustment of media. At day 3, 500 μl media was added to each well to increase the usable volume. Upon completion of each incubation, PBMCs in each sample were stained for various markers and analysed by flow cytometry ( Supplementary Table 1 ).

Samples were washed twice in eBioscience flow cytometry staining buffer (Invitrogen) and incubated in Fc-block (Human TruStain FcX, Biolegend) for 15 minutes at room temperature. After pelleting, cells were then stained for surface markers using antibodies listed in Supplementary Table 1 . Live cells were identified using LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies). After staining, cells were washed in staining buffer and fixed in Cytofix buffer (BD Biosciences) for 30 minutes (4°C) before analysis. Controls included unstained cells, as well as antibodies affixed to UltraComp eBeads Compensation Beads (Invitrogen) for compensation controls. Cells from each experiment were stained with isotype controls of each antibody, and stained heat killed cells (56°C, 15 mins) combined with live cells (1:1) were used as a live/dead control. Fluorescence minus one (FMO) controls were used to appropriately gate cell populations.

γδ T cells were isolated from activated PBMCs using EasySep Human Gamma/Delta T Cell Isolation Kit (StemCell Technologies), according to the manufacturer’s instructions. The isolated supernatant was then resuspended in supplemented RPMI1640 media before use. To confirm isolation purity, cells were analysed by flow cytometry and γδ T cells identified as CD3⁺ αβTCR^- ( Supplementary Table 1 ). This form of identification has been shown to accurately determine γδ T cells and was validated in this study as matching the cell proportion when gating with CD3⁺ Vδ1⁺ + CD3⁺ Vδ2⁺ cells ( Supplementary Figure 1 ). Isolated cells were confirmed as >90% purity before use in further experiments ( Supplementary Figure 1 ).

To determine the killing capacity of γδ T cells against Nalm6 cells, 2x10⁵ Nalm6 cells (courtesy of Qasim Rafiq, Department of Biochemical Engineering, UCL) were incubated with either 2x10⁵ or 6x10⁵ of zoledronate or OMV-activated γδ T cells. To ensure a sufficient cell number, PBMCs were activated for 14-days before isolation of γδ T cells, following the protocol described in section 2.4. To identify Nalm6 cell killing, cells from each sample were stained for expression of CD3, with Nalm6 cells identified as CD3⁺ αβTCR⁺.

The killing of SkBr3 cells (HTB-30, ATCC) was explored using the MTS assay protocol described previously by Tokuyama et al. (33). SkBr3 cells, cultured in DMEM Glutamax + 5% FBS, were seeded overnight at 37°C and 5% CO₂ in a 96-well plate at a density of 1x10⁴ cells per well. The media was then replaced with supplemented RPMI 1640 containing 14-day activated and isolated γδ T cells (with either zoledronate or OMV stimulation) at various effector to target (E:T) ratios, with media alone added as a negative control. Wells containing γδ T cells alone were used as γδ T cell controls, whilst wells with media alone were used as a blank. After 18 hours, media containing the nonadherent γδ T cells was removed and replaced with fresh media containing the MTS reagent (R&D systems). After a 3-hour incubation, optical density was measured at 490 nm and % cytotoxicity calculated as follows:

OMV immunotherapy seeks to leverage local immune-activation to facilitate tumor-cell recognition and lysis. It was therefore important to first characterise the OMV-mediated host immune response. This analysis was particularly necessary to ensure the inflammatory response was not completely abolished given the use of an OMV construct with reduced immunogenic properties.

Despite the attenuated LPS provided by ΔlpxM, E. coli MG1655 Δpal ΔlpxM OMVs stimulated PBMC to generate a robust immune response after 24 hrs. The immunogenic nature of the response was characterised by the release of pro-inflammatory cytokines IL-1β, TNFα and IL-6, as well as the anti-inflammatory cytokine IL-10 ( Figure 1 ). We also observed the production of lymphocyte-recruiting chemokines, RANTES (CCL5) and MIP-1β (CCL4).

Notably, the cytokine release profile did not indicate the presence of cytokines associated with lymphocyte activation including IL-2, IL-4, IL-5, and IL-17A. There was also no discernible effect on the release of the myeloid chemoattractant TGF-β1.

Since lymphocytes produce IL-2 once activated, it is possible that at 24 hours the quantity produced was not sufficient for detection. The incubation period was therefore extended to 5 days, with analysis focused on individual lymphocyte markers to determine cell-specific activation.

E. coli MG1655 Δpal ΔlpxM OMVs induced a broad expression of markers CD69, CD86 and CD107a across αβ T cells, γδ T cells and NK cells ( Figures 2A-C ; Supplementary Figures 2 , 3 ). Activation marker expression as a proportion of cells expressing the marker is presented in Supplementary Figures 4, 5 . The effect appeared to be concentration dependant, with a low concentration of OMVs only inducing activation marker expression in NK cells. Most notable however was the expression of CD107a, as this marker is present within lytic vesicles and displayed on the cell surface upon degranulation. Expression therefore suggests the cells were actively releasing cytotoxic factors; the release of granzyme B as well as IFN-γ was confirmed on further analysis ( Figures 2D, E ; Supplementary Figure 6 ).

Overall, activation markers and cytokines induced by OMVs suggest that the vesicles can induce an inflammatory milieu which may not only recruit lymphocytes, but additionally activate the cells to express a cytotoxic phenotype.

To investigate the effect of OMV-stimulation on the expansion of cytotoxic lymphocytes, PBMCs were stimulated with either OMVs or zoledronate. Of particular interest was the expansion of γδ T cells and NK cells, as their MHC-independent activation mechanisms provide an ability to recognize both bacterial and malignant antigens simultaneously.

In line with the widespread activation of all lymphocytes tested, there was a dramatic expansion of the total immune cell population in response to OMVs. The PBMCs were seen to reach a similar total concentration across all conditions ( Supplementary Figure 7 ), potentially due to limited resources inhibiting further growth.

The proportion of αβ T cells remained constant upon OMV activation, relative to the pre-stimulated control ( Figure 3A ; Supplementary Figure 8 ). In contrast, OMV + IL-2 activation induced γδ T cells to expand to around 35% of the total cell population, significantly greater than the ~7% achieved with IL-2 alone ( Figure 3B ). This expansion was similar to that observed in response to zoledronate + IL-2, a compound utilized for the specific expansion of the Vγ9Vδ2 (Vδ2⁺) subtype of γδ T cells. Whilst it is interesting to note that IL-2 alone facilitated the growth of the Vδ1⁺ subtype within the γδ T cell population, we observed a dominance in the proportion of Vγ9Vδ2 T cells in response to both zoledronate and OMV activation ( Figure 3D ).

Since the overall cell number was similar to that seen with IL-2 alone, it appears that the proliferation of Vγ9Vδ2 T cells in response to OMVs was mostly at the expense of NK cell expansion ( Figure 3C ). Their relative proportion in the experimental cell milieu was not significantly different compared to the starting population, though there was an apparent phenotypic shift to the immunoregulatory CD56^bright subtype. Given their similarity in proportion however, it is likely that this preference to CD56^bright cells was driven at least in part through the effect of IL-2 supplementation ( Figure 3E ).

Though it was apparent that γδ T cells respond to OMVs, it was necessary to confirm their potential oncolytic activity despite the microbial means of activation. Isolated γδ T cells were expanded with either OMVs or zoledronate, and their killing capacity determined against a leukaemic (Nalm6) and breast cancer (SkBr3) cell line. Indeed, OMV-activated γδ T cells were able to effectively initiate cell killing ( Figure 4 ). Whilst inter-donor variability meant Nalm6 killing at a 1:1 effector to target (E:T) ratio did not reach statistical significance, the oncolytic capacity of OMV-expanded cells was equivalent to that of cells activated with zoledronate. This killing effect was also observed in SkBr3, again not significantly different to that of cells activated with zoledronate ( Figure 4 ).