Study of the immune microenvironment in Mycosis Fungoides

Mycosis fungoides is a rare, indolent and incurable lymphoma of T cells affecting the skin. While the majority of patients have survival measured in the decades, around one third present with advanced disease and another quarter progress rapidly. Advanced disease compromises skin barrier integrity, and is amenable to few effective treatments. The malignant cell is usually a CD4+ T cell, which in healthy individuals plays a key role in our protection against pathogens and cancer. As a clonal expansion of an existing T cell, these tumour cells already possess an idiomatic mutation in the form of the rearranged T cell receptor (TCR) gene. Paired skin biopsies and blood was taken from consenting patients, the skin sample digested, and stained with anti-T Cell Receptor V-beta region (TCR-Vβ) antibodies to identify a clonotypic tumour population. This antibody was used to discriminate tumour and Tumour Infiltrating Lymphocytes (TIL) in a study looking at immune checkpoint markers, functionality, cytotoxicity and regulatory phenotypes. Single cell RNA sequencing (scRNA-seq) was employed with simultaneous TCR sequencing. We found that TIL demonstrated a homogeneous exhaustion phenotype which was consistent between samples and with disease progression. The tumour population was heterogeneous, clustering into three phenotypic groups, and demonstrating high polyfunctionality, overproducing IL-4 and IL-17a, in what is likely immune evasion by deviation. The tumour did not show a suppressive phenotype, and a mathematical model was created to understand why this may not provide an advantage to the tumour. scRNA-seq analysis found several recurrent differentially expressed genes in tumour, which may represent promising targets for therapeutic potential. This work is the largest single cell study of the immune microenvironment in mycosis fungoides, and utilises two novel methods to discriminate tumour and TIL, adding substantially to our understanding of the aetiology and progression of mycosis fungoides. Developing new therapeutic targets in mycosis fungoides must ensure that the infiltrating T cells are retained while targeting the tumour population, and this work identifies several avenues by which this could be achieved