Fabio Ticconi

Regulatory T cells (T _regs ) are present in lymphoid and nonlymphoid tissues where they restrict immune activation, prevent autoimmunity, and regulate inflammation. T _regs in nonlymphoid tissues are typically resident, whereas those in lymph nodes (LNs) are considered to recirculate. However, T _regs in LNs are not a homogenous population, and circulation kinetics of different T _reg subsets are poorly characterized. Furthermore, whether T _regs can acquire memory T cell properties and persist for extended periods after their activation in LNs is unclear. Here, we used in situ labeling with a stabilized photoconvertible protein to uncover turnover rates of T _regs in LNs in vivo. We found that, whereas most T _regs in LNs recirculate, 10 to 20% are memory-like resident cells that remain in their respective LNs for weeks to months. Single-cell RNA sequencing revealed that LN-resident cells are a functionally and ontogenetically heterogeneous population and share the same core residency gene signature with conventional CD4 ⁺ and CD8 ⁺ T cells. Resident cells in LNs did not actively proliferate and did not require continuous T cell receptor (TCR) signaling for their residency. However, resident and circulating T _regs had distinct TCR repertoires, and each LN contained exclusive clonal subpopulations of resident T _regs . Our results demonstrate that, similar to conventional T cells, T _regs can form resident memory-like populations in LNs after adaptive immune responses. Specific and local suppression of immune responses by resident T _regs in draining LNs might provide previously unidentified therapeutic opportunities for the treatment of local chronic inflammatory conditions.

Introduction: SARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants.Methods: We combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8⁺ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19.Results: We observed increased CD8⁺ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8⁺ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8⁺ T cells revealed heterogeneity among CD16⁺ NK-like CD8⁺ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions.Discussion: We propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8⁺ effector T cells, ultimately resulting in the appearance of NK-like CD8⁺ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8⁺ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8⁺ T cells in COVID-19 severity.

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSCs) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, we performed a systematic comparison on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single-clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1−/+ multipotent progenitors showed a proproliferative gene signature and HSCs showed a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1−/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. We therefore propose a crucial role for Csnk1a1 haploinsufficiency in the selective advantage of 5q-HSCs, implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer.

The current COVID-19 pandemic represents a global challenge. A better understanding of the immune response against SARS-CoV-2 is key to unveil the differences in disease severity and to develop future vaccines targeting novel SARS-CoV-2 variants. Feature barcode technology combined with CITE-seq antibodies and DNA-barcoded peptide-MHC I Dextramer reagents enabled us to identify relevant SARS-CoV-2-derived epitopes and compare epitope-specific CD8 ⁺ T cell populations between mild and severe COVID-19. We identified a strong CD8 ⁺ T cell response against an S protein-derived epitope. CD8 ⁺ effector cells in severe COVID-19 displayed hyperactivation, T cell exhaustion and were missing characteristics of long-lived memory T cells. We identify A*0101 WTAGAAAYY as an immunogenic CD8 ⁺ T cell epitope with the ability to drive clonal expansion. We provide an in-depth characterization of the CD8 ⁺ T cell-mediated response to SARS-CoV-2 infection which will be relevant for the development of molecular and targeted therapies and potential adjustments of vaccination strategies.

De novo DNA methyltransferase 3A (DNMT3A) plays pivotal roles in hematopoietic differentiation. In this study, we followed the hypothesis that alternative splicing ofDNMT3Ahas characteristic epigenetic and functional sequels. SpecificDNMT3Atranscripts were either down-regulated or overexpressed in human hematopoietic stem and progenitor cells, and this resulted in complementary and transcript-specific DNA methylation and gene expression changes. Functional analysis indicated that, particularly, transcript 2 (coding for DNMT3A2) activates proliferation and induces loss of a primitive immunophenotype, whereas transcript 4 interferes with colony formation of the erythroid lineage. Notably, in acute myeloid leukemia expression of transcript 2 correlates with its in vitro DNA methylation and gene expression signatures and is associated with overall survival, indicating thatDNMT3Avariants also affect malignancies. Our results demonstrate that specificDNMT3Avariants have a distinct epigenetic and functional impact. Particularly, DNMT3A2 triggers hematopoietic differentiation and the corresponding signatures are reflected in acute myeloid leukemia.

Significance: The two E-type cyclins E1 and E2 are known to interact with Cdk2 and are thought to trigger cell cycle activity in carcinogenesis. However, the individual contributions of cyclin E1, cyclin E2, and Cdk2 for initiation and progression of hepatocellular carcinoma (HCC) are unknown. In the present study, we discovered that only cyclin E1—but not cyclin E2—is essential for initiation of liver cancer and requires Cdk2. Unexpectedly, advanced liver cancer progression can be mediated in presence of any E-cyclin, but in a Cdk2-independent manner. We identified the specific expression profiles of cyclin E1-dependent and cyclin E1-independent hepatoma cells. These signatures are useful for predicting patient prognosis and for developing novel cyclin E-based HCC therapies.

Background: The term systemic juvenile idiopathic arthritis (sJIA) describes an autoinflammatory condition characterized by arthritis and severe systemic inflammation, which in later stages can transform into interleukin (IL)-17-driven autoimmune arthritis. IL-1 antagonists have been used with good efficacy in the early stages of sJIA. Methods: A whole transcriptome analysis of peripheral blood RNA samples was performed in six patients with sJIA and active systemic disease, before initiating treatment with the IL-1 beta receptor antagonist anakinra, and after induction of inactive disease, compared with a single-sample control cohort of 21 patients in several clinical stages of sJIA activity. Whole transcriptomes were compared longitudinally and interindividually including gene ontology and motif enrichment analysis of differentially expressed genes. Results: There were 741 transcripts were identified using a threshold with a p value 2. HLADRB1 and CD74 were identified as the most strongly upregulated genes in inactive compared to active disease; CD177 expression was significantly enhanced in active disease compared to inactive disease. Motif enrichment analysis revealed STAT4, BCL6, and STAT3 as the most prominent transcription factors that were present during active disease. In addition, strong upregulation of the major histocompatability complex II (MHCII) ligand CD74 was found in both active and inactive sJIA compared to healthy controls. Conclusion: Using transcription factor motif enrichment, this study identifies novel putative pathways in sJIA (STAT4, BCL6) implicating B cell activation at an earlier stage than predicted in refractory disease. The implication of BCL-6 dependent pathways argues for occurrence of autoimmunity early within the process of sJIA chronification. Transcriptional regulation of HLA-DRB1, a recently described independent genetic risk factor, in combination with its cooperating partner CD74 in patients where sJIA is confirmed, supports pathogenic involvement in alterations in antigen presentation during sJIA.