Investigating cutaneous T-cell trafficking during treatment with immune checkpoint blockade

Background Antibodies that block the immune checkpoint response proteins PD1 and CTLA4 have revolutionised the treatment of metastatic melanoma. There is substantial clinical heterogeneity in the treatment response however, with many patients not benefitting.

Recent work has demonstrated that response to immune checkpoint blockade (ICB) is associated with increased clonal expansion of T cells in the peripheral blood. The degree to which these peripherally expanded T cell clones migrate into tumour and other tissues remains unclear.

Aims The skin and peripheral blood provide easily accessible sites to evaluate the trafficking of immune cells following ICB therapy.

This project aims to: (i) Examine the overlap between T cell clones in the skin and peripheral blood at baseline and post-ICB therapy in patients with metastatic melanoma using bulk T cell receptor (TCR) sequencing. (ii) Compare phenotypic changes within the T cell clones in the skin and peripheral blood following ICB therapy, at the single cell level.

Methods Patients with metastatic melanoma treated with ICB therapy will be recruited via a specialised melanoma clinic in Oxford.

Biopsies of non-cancerous skin and peripheral blood samples will be obtained from 12 individuals at baseline and post-cycle 1 of ICB therapy. Bulk TCR sequencing will be performed on the samples obtained from all 12 individuals.

This will enable comparison of the baseline overlap in T cell clones within the skin and blood and assessment of the flux between these compartments with treatment.

Single cell sequencing of T cells infiltrating the skin and blood from 4 individuals will be performed pre and post treatment using 10x Genomics Chromium kits.

Skin and blood samples from the same individual will be multiplexed onto the same chip to save costs and minimise batch effects, with anti-CD45 antibodies conjugated to different nucleic acid ‘barcodes’ used to enable post-sequencing dissection of tissue origin.

Using established bioinformatic pipelines, the gene expression across different T cell clones in the skin and blood at baseline and post-ICB therapy will be examined.

Statistical methods will be applied to determine overlap between tissues, and the significance of changes in gene expression and clone size, with a view to integrating findings with clinical otucomes.

How the results of this research will be used The results of this research will provide insights into the dynamics of T cell trafficking in the treatment of cancer with immunotherapy, shedding light onto clinical heterogeneity and aiding the application of stratified medicine.