Originally discovered as an inducer of apoptosis, the TNF-family receptor Fas (CD95, APO-1, TNFRSF6) has more recently been found to have functions beyond cell death, including T cell co-stimulation and promoting terminal differentiation of CD4 and CD8 T cells. Other TNF family members also discovered as apoptosis inducers, such as TRAIL (APO-2L, TNFSF10), can promote inflammation through caspase-8. Surprisingly, non-apoptotic signaling through Fas can protect from the autoimmunity seen in Fas deficiency independently from the cell death inducing functions of the receptor. Non-apoptotic Fas signaling can induce tumor cell growth and migration, and impair the efficacy of T cell adoptive immunotherapy. Blocking of non-apoptotic functions of these receptors may be a novel strategy to regulate autoimmunity and inflammation, and enhance antitumor immunity.

T-cell landscape differences between cutaneous squamous cell carcinoma (cSCC) tumors in immune competent (SCC in IC) and immunocompromised organ transplant recipients (TSCC in OTR) are unclear. We developed an analytical method to define tumor infiltrating lymphocyte (TIL) phenotype in cSCC from immune competent and immune suppressed patients using single-cell TCR sequencing and gene expression data. TSCC exhibits reduced proportions of cytotoxic and naïve TILs and similar numbers of regulatory TILs. Fewer, more heterogeneous TCR clonotypes are observed in TIL from OTR. Most TCR sequences for top ten clonotypes correspond to known antigens, while 24% correspond to putative neoantigens. OTR show increased cSCC events over 12 months possibly due to reduced cytotoxic T-cells. Our novel method of barcoding CD8+ T-cells is the first providing gene expression and TCR sequences in cSCC. Knowledge regarding putative antigens recognized by TCRs with phenotypic function of T-cells bearing those TCRs could facilitate personalized cSCC treatments.

LINE-1 retrotransposons are sequences capable of copying themselves to new genomic loci via an RNA intermediate. New studies implicate LINE-1 in a range of diseases, especially in the context of aging, but without an accurate understanding of where and when LINE-1 is expressed, a full accounting of its role in health and disease is not possible. We therefore developed a method—5′ scL1seq—that makes use of a widely available library preparation method (10x Genomics 5′ single cell RNA-seq) to measure LINE-1 expression in tens of thousands of single cells. We recapitulated the known pattern of LINE-1 expression in tumors—present in cancer cells, absent from immune cells—and identified hitherto undescribed LINE-1 expression in human epithelial cells and mouse hippocampal neurons. In both cases, we saw a modest increase with age, supporting recent research connecting LINE-1 to age related diseases.

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas–Fas and Fas–FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.