64P - Gene-editing of T cells to provide resistance against macrophage-mediated suppression: setting up an in vitro model

Background

Tumor associated macrophages (TAMs) are highly abundant immune cells present within the microenvironment of multiple tumor types. TAMs with a type 2 (M2)-like phenotype generally associate with poor prognosis and resistance against immunotherapy. In fact, we observed high frequencies of M2-like TAMs relative to those of CD8⁺ T cells according to gene expression analyses in multiple tumor types. In this study, we aim to engineer CD8⁺ T cells that resist M2-like macrophage-mediated suppression.

Methods

First, we have set up an in vitro co-culture model of human autologous M2-like macrophages and CD8⁺ T cells that yields T cell suppression. To this end, we have polarized in vitro monocytes from healthy donors into M2-like macrophages using a cytokine cocktail, after which we have optimized critical parameters, such as type of T cell stimulation, co-culture duration and the ratio between the two cell types.

Results

With this model, we have demonstrated that M2-like macrophages from multiple donors, when co-cultured with anti-CD3/CD28 antibody-stimulated autologous CD8⁺ T cells for 6 days, suppressed T cell proliferation by 50%. In a second step, we have adapted this model to a CRISPR-Cas9 gene editing-based approach: single guide RNA lentiviral infection with Cas9 electroporation (SLICE). We have successfully performed single gene editing, and are currently editing CD8⁺ T cells with gene libraries targeting kinases.

Conclusions

Taken together, we present a robust in vitro model of M2-like macrophage-mediated suppression of CD8⁺ T cells which enables the identification of genes that contribute to suppression-resistant CD8⁺ T cells. The making of such synthetic T cells is anticipated to aid the anti-tumor efficacy of adoptive T cell therapy.

Legal entity responsible for the study

Erasmus MC.

Funding

KWF Kankerbestrijding.

Disclosure

All authors have declared no conflicts of interest.