Nalini Srinivas ^1,2 Lukas Peiffer ^1,3 Kai Horny ¹ Kuan Cheok Lei ¹ Terkild B. Buus ³ Linda Kubat ¹ Meng Luo ¹ Menghong Yin ¹ Ivelina Spassova ^1,2 Antje Sucker ² Farnoush Farahpour ^4,5 Jan Kehrmann ⁶ Selma Ugurel ² Elisabeth Livingstone ² Thilo Gambichler ⁷ Niels Odum ³ Juergen C. Becker ^1,2*

• ¹ Institute for Translational Skin Cancer Research, German Cancer Consortium, German Cancer Research Center (DKFZ), Essen, North Rhine-Westphalia, Germany
• ² Clinic for Dermatology, University Hospital of Essen, Essen, North Rhine-Westphalia, Germany
• ³ Department of Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Capital Region of Denmark, Denmark
• ⁴ Bioinformatics and Computational Biophysics, University Duisburg-Essen, Essen, Germany
• ⁵ Group of Molecular Cell biology, Institute for Cell Biology (Cancer Research), University Hospital Essen, Essen, Germany
• ⁶ Institute for Medical Microbiology, University Hospital Essen, Essen, North Rhine-Westphalia, Germany
• ⁷ Department of Dermatology, Venereology and Allergology, St. Josef Hospital, Bochum, North Rhine-Westphalia, Germany

Background: Mycosis fungoides (MF) is the most common subtype of cutaneous T-cell lymphoma (CTCL). Comprehensive analysis of MF cells in situ and ex vivo is complicated by the fact that is challenging to distinguish malignant from reactive T cells with certainty.To overcome this limitation, we performed combined single-cell RNA (scRNAseq) and Tcell receptor TCR sequencing (scTCRseq) of skin lesions of cutaneous MF lesions from 12 patients. A sufficient quantity of living T cells was obtained from 9 patients, but 2 had to be excluded due to unclear diagnoses (coexisting CLL or revision to a fixed toxic drug eruption).From the remaining patients we established single-cell mRNA expression profiles and the corresponding TCR repertoire of 18,630 T cells. TCR clonality unequivocally identified 13,592 malignant T cells. Reactive T cells of all patients clustered together, while malignant cells of each patient formed a unique cluster expressing genes typical of naive/memory, such as CD27, CCR7 and IL7R, or cytotoxic T cells, e.g., GZMA, NKG7 and GNLY. Genes encoding classic CTCL markers were not detected in all clusters, consistent with the fact that mRNA expression does not correlate linearly with protein expression. Nevertheless, we successfully pinpointed distinctive gene signatures differentiating reactive malignant from malignant T cells: keratins (KRT81, KRT86), galectins (LGALS1, LGALS3) and S100 genes (S100A4, S100A6) being overexpressed in malignant cells.Conclusions: Combined scRNAseq and scTCRseq not only allows unambiguous identification of MF cells, but also revealed marked heterogeneity between and within patients with unexpected functional phenotypes. While the correlation between mRNA and protein abundance was limited with respect to established MF markers, we were able to identify a single-cell gene expression signature that distinguishes malignant from reactive T cells.