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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., Methods: To overcome this limitation, we performed combined single-cell RNA (scRNAseq) and T-cell 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)., Results: 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., Competing Interests: SU declares research support from Bristol Myers Squibb and Merck Serono; speakers and advisory board honoraria from Bristol Myers Squibb, Merck Sharp and Novartis, and travel support from Bristol Myers Squibb, Merck Sharp & Dohme and Pierre Fabre. EL reports personal fees, honoraria, and support for attending meetings or travel grants from Bristol Myers Squibb, Medac, Novartis, Pierre Fabre, and Sun Pharma, and honoraria from MSD, Recordati, and Sanofi. EL participated on a drug safety monitoring or advisory board for Bristol Myers Squibb, Novartis, Sanofi, and Sun Pharma. TG has received speakers and/or advisory board honoraria from BMS, Sano-fi-Genzyme, MSD, Novartis Pharma, Roche, Abbvie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, Merck-Serono, outside the submitted work. JB is receiving speaker’s bureau honoraria from Amgen, Pfizer, Recordati and Sanofi, and is a paid consultant/advisory board member/DSMB member for Almirall, Boehringer Ingelheim, InProTher, ICON, Merck Serono, Pfizer, 4SC and Sanofi/Regeneron. His group receives research grants from Bristol Myers Squibb, Merck Serono, HTG, IQVIA and Alcedis GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Srinivas, Peiffer, Horny, Lei, Buus, Kubat, Luo, Yin, Spassova, Sucker, Farahpour, Kehrmann, Ugurel, Livingstone, Gambichler, Ødum and Becker.)

Background: Sézary Syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL). In SS patients, malignant T cells are circulating through the blood and cause erythroderma., Objective: To compare the transcriptome of single cells in blood and skin samples from a patient with advanced SS., Methods: We utilized combined single cell RNA and T-cell receptor (TCR) sequencing (scRNA-seq)., Results: We scrutinized the malignant T cells in blood and skin in an unbiased manner without pre-sorting of cells. We observed different phenotypes of the same monoclonal malignant T-cell population, confirmed by TCR sequencing and inferred copy number variation analysis. Malignant T cells present in the circulating blood expressed genes resembling central memory T cells such as CCR7 , IL7R and CD27 . In the skin, we detected two major malignant T-cell populations: One subpopulation was closely related to the malignant T cells from the blood, while the other subpopulation expressed genes reminiscent of skin resident effector memory T cells including GZMB and NKG7 . Pseudotime analysis indicated crucial transcriptomic changes in the transition of malignant T cells between blood and skin. These changes included the differential regulation of TXNIP , a putative tumor suppressor in CTCL, and the adaptation to the hypoxic conditions in the skin. Tumor cell proliferation in the skin was supported by stimulating interactions between myeloid cells and malignant T cells., Conclusions: Using scRNA-seq we detected a high degree of functional heterogeneity within the malignant T-cell population in SS and highlighted crucial differences between SS cells in blood and skin., Competing Interests: TG has received speakers and/or advisory board honoraria from BMS, Sanofi, MSD, Novartis Pharma, Janssen, Roche, Sun-Pharma, Abbvie, Almirall, Janssen, Lilly, Pfizer, Pierre Fabre, Merck-Serono, outside the submitted work. RS has received speakers and/or honoraria from Kyowa Kirin, Takeda, Novartis and Recordati Rare Diseases. LS has received speakers and/or advisory board honoraria from BMS, Sun-Pharma, MSD, and Novartis. JB is receiving speaker's bureau honoraria from Amgen, Pfizer, Recordati and Sanofi, is a paid consultant/advisory board member/DSMB member for Almirall, Boehringer Ingelheim, InProTher, ICON, Merck-Serono, Pfizer, 4SC, and Sanofi. His group receives research grants from Merck Serono, HTG, IQVIA, and Alcedis. All other authors have no conflict of interest to declare., (Copyright © 2023 Peiffer, Gambichler, Buus, Horny, Gravemeyer, Furtmann, Spassova, Kubat, Susok, Stranzenbach, Srinivas, Ødum and Becker.)

The active form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), mediates its immunomodulatory effects by binding to the vitamin D receptor (VDR). Here, we describe a new point mutation in the DNA-binding domain of the VDR and its consequences for 1,25(OH) 2 D 3 signaling in T cells from heterozygous and homozygous carriers of the mutation. The mutation did not affect the overall structure or the ability of the VDR to bind 1,25(OH) 2 D 3 and the retinoid X receptor. However, the subcellular localization of the VDR was strongly affected and the transcriptional activity was abolished by the mutation. In heterozygous carriers of the mutation, 1,25(OH) 2 D 3 -induced gene regulation was reduced by ~ 50% indicating that the expression level of wild-type VDR determines 1,25(OH) 2 D 3 responsiveness in T cells. We show that vitamin D-mediated suppression of vitamin A-induced gene regulation depends on an intact ability of the VDR to bind DNA. Furthermore, we demonstrate that vitamin A inhibits 1,25(OH) 2 D 3 -induced translocation of the VDR to the nucleus and 1,25(OH) 2 D 3 -induced up-regulation of CYP24A1. Taken together, this study unravels novel aspects of vitamin D signaling and function of the VDR in human T cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Al-Jaberi, Kongsbak-Wismann, Aguayo-Orozco, Krogh, Buus, Lopez, Rode, Gravesen, Olgaard, Brunak, Woetmann, Ødum, Bonefeld and Geisler.)