Code to reproduce figures in the paper is available here: github.com/callumnattre...

PDO scRNA-seq data can be found here: zenodo.org/records/1582...

All single-cell PTM files from γδ T cells and PDOs can be found here: community.cytobank.org/cytobank/pro...

This project was an experimental tour de force from Callum Nattress, Rhianna O'sullivan, Daniel Fowler, Colin Hutton, Petra Vlckova, @vivianlilab.bsky.social, Kerry Chester, John Anderson, @marta-barisa.bsky.social, and Jon Fisher. Thanks to @cruk-cityoflondon.bsky.social for funding. (13/13)

In summary, these results suggest that γδ T cell multimodal cytotoxicity can buffer γδ T cell signalling from patient-specific cancer cell immunomodulation, allowing γδ T cells from a range of donors to kill chemorefractory cancer cells. (12/13)

This demonstrates that these lethal, chemoresistant revCSCs cancer cells are therapeutically targetable by cellular therapies. (11/13)

CRC PDOs comprise an admixture of chemosensitive proliferative colonic stem cells (proCSCs) and chemorefractory revival colonic stem cells (revCSCs). Crucially, stIL-15-γδ T cells can kill PDOs irrespective of stem cell admixture — including chemorefractory PDOs. (10/13)

To confirm that the anti-B7-H3 antibody was only inducing ADCC and not invoking the putative immune checkpoint properties of B7-H3, we also demonstrated that only a complete IgG — and not an Fc-null IgG — could trigger multimodal killing. (9/13)

Importantly, PDO immunomodulation of γδ T cells correlates with reduce AIC killing (i.e. the more PDOs change γδ T cell signalling, the worse γδ T cells kill). However, when γδ T cells also use AIC + ADCC, PTM signalling was restored and γδ T cells could kill all PDOs. (8/13)

We found that γδ T cells can kill both MSS and MSI CRC PDOs. However, when γδ T cells only use AIC, γδ T cell signalling converges on the target PDO, irrespective of T-cell donor. This suggests that γδ T cells are immunomodulated by PDOs and ITH is dominant over IDH. (7/13)

To investigate how γδ T cells from various T-cell donors (IDH) kill cancer cells from a range of CRC patients (ITH) via AIC and ADCC, we performed single-cell signalling analysis of γδ T cells from x4 donors co-cultured with x10 CRC PDOs, +/- B7-H3 mAb (x780 3D cultures). (6/13)

Next, we found that stIL15-engineered γδ T cells can rapidly kill MSI colorectal cancer (CRC) patient-derived organoids (PDOs) in 3D via AIC. Killing could be further increased by inducing ADCC via a novel anti-B7-H3 antibody — demonstrating multimodal cytotoxicity. (5/13)

First, we found that expressing stabilised IL-15Rα–IL-15 (stIL15) enables γδ T cells to survive in 3D cultures without serum or cytokine support. This is important because many T-cell killing assays are performed in 2D with serum, but tumours are 3D and nutrient-deprived. (4/13)

To explore this, we performed a systematic single-cell phenoscaping study of >1,000 organoid cultures testing the combinatorial interactions between γδ T cell killing modalities (AIC vs ADCC), γδ T cell inter-donor heterogeneity, and cancer cell inter-tumour heterogeneity. (3/13)

γδ T cells can kill cancer cells via antibody-independent cytotoxicity (AIC) and antibody-dependent cellular cytotoxicity (ADCC), but how this killing varies across T cell donors (inter-donor heterogeneity, IDH) and patients (inter-tumour heterogeneity, ITH) is unclear. (2/13)

Yes!!!