Single-cell RNA sequencing comparison of CD4+, CD8+ and T-cell receptor γδ+ cutaneous T-cell lymphomas reveals subset-specific molecular phenotypes

Background Malignant clones of primary cutaneous T-cell lymphomas (CTCL) can show a CD4(+), CD8(+) or T-cell receptor (TCR)-gamma delta(+) phenotype, but their individual impact on tumour biology and skin lesion formation remains ill defined. Objectives To perform a comprehensive molecular characterization of CD4(+) vs. CD8(+) and TCR-gamma delta(+) CTCL lesions. Methods We performed single-cell RNA sequencing (scRNAseq) of 18 CTCL skin biopsies to compare classic CD4(+) advanced-stage mycosis fungoides (MF) with TCR-gamma/delta(+) MF and primary cutaneous CD8(+) aggressive epidermotropic cytotoxic T-cell lymphoma (Berti lymphoma). Results Malignant clones of TCR-gamma/delta(+) MF and Bertilymphoma showed similar clustering patterns distinct from CD4(+) MF, along with increased expression of cytotoxic markers such as NKG7, CTSW, GZMA and GZMM. Only advanced-stage CD4(+) MF clones expressed central memory T-cell markers (SELL, CCR7, LEF1), alongside B1/B2 blood involvement, whereas TCR-gamma delta(+) MF and Berti lymphoma harboured a more tissue-resident phenotype (CD69, CXCR4, NR4A1) without detectable cells in the blood. CD4(+) MF and TCR-gamma delta(+) MF skin lesions harboured strong type 2 immune activation across myeloid cells, while Berti lymphoma was more skewed toward type 1 immune responses. Both CD4(+) MF and TCR-gamma delta(+) MF lesions showed upregulation of keratinocyte hyperactivation markers such as S100A genes and KRT16. This increase was entirely absent in Berti lymphoma, possibly reflecting an aberrant keratinocyte response to invading tumour cells, which could contribute to the formation of the typical ulceronecrotic lesions within this entity. Conclusions Our scRNAseq profiling study reveals specific molecular patterns associated with distinct CTCL subtypes. Cutaneous T-cell lymphomas are types of skin cancer characterized by a rapid increase in a type of white blood cell called 'T lymphocytes'. They consist of a similar group of diseases, many of which are still poorly understood. Cancerous T lymphocytes in cutaneous lymphomas express the same T-cell receptor (TCR) sequence, hence the term 'clones' is often used to describe these malignant cells. Usually, these clonal T lymphocytes express an alpha/beta TCR in conjunction with protein called CD4. In rare cases, they may express an alpha/beta TCR along with a different protein called CD8, or instead display a gamma/delta TCR. To better understand these diseases, we compared three major subsets of cutaneous lymphomas (CD4(+), CD8(+), and TCR-gamma/delta) on a molecular level. We found specific signatures associated with each of the diseases, which may help with future strategies to better treat people with cutaneous T-cell lymphomas. What is already known about this topic? center dot Malignant clones of primary cutaneous T-cell lymphomas can show CD4, CD8 or T-cell receptor (TCR)-gamma delta phenotypes, but their individual pathophysiological impact remains ill defined. What does this study add? center dot Using single-cell RNA sequencing, we found that malignant clones of advanced-stage CD4(+) mycosis fungoides (MF) lesions harboured a central memory phenotype with concomitant expression of the therapeutic targets CCR4 and KIR3DL2, whereas malignant clones of TCR-gamma/delta(+) MF and primary cutaneous aggressive epidermotropic CD8(+) T-cell lymphoma (Berti lymphoma) showed a tissue-resident memory signature. center dot While keratinocyte activation markers and skin defence molecules were highly upregulated in both CD4(+) MF and TCR-gamma delta(+) MF, this was not seen in Berti lymphoma, potentially contributing to its characteristic ulceronecrotic clinical presentation. What is the translational message? center dot We found characteristic molecular markers differentiating malignant clones and the tumour microenvironment of CD4(+), CD8(+) and TCR-gamma delta(+) primary cutaneous lymphomas that might help to facilitate future targeted treatment approaches.