■真菌病(MF)是皮肤T细胞淋巴瘤(CTCL)最常见的亚型。对MF细胞的原位和离体的综合分析由于以下事实而变得复杂,即确定性地区分恶性和反应性T细胞是具有挑战性的。
■为了克服这一限制,我们对12例患者皮肤MF病变的皮肤病变进行了联合单细胞RNA(scRNAseq)和T细胞受体TCR测序(scTCRseq).从9名患者中获得了足够数量的活T细胞,但2例由于诊断不明确(同时存在CLL或修正为固定的毒性药疹)而不得不排除.
■从其余患者中,我们建立了18,630个T细胞的单细胞mRNA表达谱和相应的TCR库。TCR克隆性明确鉴定了13,592个恶性T细胞。所有患者的反应性T细胞聚集在一起,而每个患者的恶性细胞形成了一个独特的簇,表达典型的幼稚/记忆基因,如CD27、CCR7和IL7R,或者细胞毒性T细胞,例如,GZMA,NKG7和GNLY。编码经典CTCL标记的基因未在所有簇中检测到,与mRNA表达与蛋白质表达不线性相关的事实一致。然而,我们成功地确定了区分反应性恶性和恶性T细胞的独特基因特征:角蛋白(KRT81,KRT86),galectin(LGALS1,LGALS3)和S100基因(S100A4,S100A6)在恶性细胞中过表达。
■组合的scRNAseq和scTCRseq不仅允许明确识别MF细胞,但也揭示了具有意外功能表型的患者之间和内部的明显异质性。虽然mRNA和蛋白质丰度之间的相关性相对于已建立的MF标记是有限的,我们能够鉴定出区分恶性T细胞和反应性T细胞的单细胞基因表达特征.
UNASSIGNED: 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.
UNASSIGNED: 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).
UNASSIGNED: 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.
UNASSIGNED: 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.