Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Differentiation of Peripheral Treg.

This chapter shows protocols for the differentiation of peripheral Treg (pTreg) from polyclonal and monoclonal CD4+ T cells. Polyclonal naïve CD4+ T cells can differentiate into pTreg upon adoptive transfer into Foxp3-diphtheria toxin receptor transgenic recipient mice in which endogenous Tregs are transiently depleted by administration of diphtheria toxin before adoptive transfer. Differentiation of monoclonal pTreg is induced through oral delivery of ovalbumin into RAG-deficient DO11.10 mice, in which T cells are ovalbumin specific. We show the isolation of naïve CD4+ T cells by flow cytometry, the administration of ovalbumin in drinking water, and the analysis tools, including an optional protocol for the enrichment of analysis samples in CD4+ T cells using a magnetic purification.

Interruption of Thymic Activity in Adult Mice Improves Responses to Tumor Immunotherapy.

The thymus produces precursors of both conventional T cells (Tconv; also known as effector T cells) and regulatory T cells (Treg) whose interactions prevent autoimmunity while allowing efficient protective immune responses. Tumors express a composite of self-antigens and tumor-specific Ags and engage both Tconv and Treg. Along the aging process, the thymus involutes, and tumor prevalence increases, a correlation proposed previously to result from effector cell decline. In this work, we directly tested whether interruption of thymic activity in adult mice affects Foxp3-expressing Treg composition and function and alters tumor immune surveillance. Young adult mice, on two different genetic backgrounds, were surgically thymectomized (TxT) and analyzed or challenged 2 mo later. Cellular analysis revealed a 10-fold decrease in both Tconv and Treg numbers and a bias for activated cells. The persisting Treg displayed reduced stability of Foxp3 expression and, as a population, showed a compromised return to homeostasis upon induced perturbations. We next tested the growth of three tumor models from different tissue origins and/or presenting distinct degrees of spontaneous immunogenicity. In none of these conditions, adult TxT facilitated tumor growth. Rather, TxT enhanced the efficacy of antitumor immunotherapies targeting Treg and/or the immune checkpoint CTLA4, as evidenced by the increased frequency of responder mice and decreased intratumoral Treg to CD8+IFN-γ+ cell ratio. Together, our findings point to a scenario in which abrogation of thymic activities affects preferentially the regulatory over the ridding arm of the immune activities elicited by tumors and argues that higher prevalence of tumors with age cannot be solely attributed to thymic output decline.

T-cell dysregulation in COVID-19.

T-cells play key roles in immunity to COVID-19 as well as the development of severe disease. T-cell immunity to COVID-19 is mediated through differentiated CD4+ T-cells and cytotoxic CD8+ T-cells, although their differentiation is often atypical and ambiguous in COVID-19 and single cell dynamics of key genes need to be characterized. Notably, T-cells are dysregulated in severe COVID-19 patients, although their molecular features are still yet to be fully revealed. Importantly, it is not clear which T-cell activities are beneficial and protective and which ones can contribute to the development of severe COVID-19. In this article, we examine the latest evidence and discuss the key features of T-cell responses in COVID-19, showing how T-cells are dysregulated in severe COVID-19 patients. Particularly, we highlight the impairment of FOXP3 induction in CD4+ T-cells and how the impaired FOXP3 expression can lead to the differentiation of abnormally activated (hyperactivated) T-cells and the dysregulated T-cell responses in severe patients. Furthermore, we characterise the feature of hyperactivated T-cells, showing their potential contribution to T-cell dysregulation and immune-mediated tissue destruction (immunopathology) in COVID-19.

T-Cell Hyperactivation and Paralysis in Severe COVID-19 Infection Revealed by Single-Cell Analysis.

Severe COVID-19 patients show various immunological abnormalities including T-cell reduction and cytokine release syndrome, which can be fatal and is a major concern of the pandemic. However, it is poorly understood how T-cell dysregulation can contribute to the pathogenesis of severe COVID-19. Here we show single cell-level mechanisms for T-cell dysregulation in severe COVID-19, demonstrating new pathogenetic mechanisms of T-cell activation and differentiation underlying severe COVID-19. By in silico sorting CD4+ T-cells from a single cell RNA-seq dataset, we found that CD4+ T-cells were highly activated and showed unique differentiation pathways in the lung of severe COVID-19 patients. Notably, those T-cells in severe COVID-19 patients highly expressed immunoregulatory receptors and CD25, whilst repressing the expression of FOXP3. Furthermore, we show that CD25+ hyperactivated T-cells differentiate into multiple helper T-cell lineages, showing multifaceted effector T-cells with Th1 and Th2 characteristics. Lastly, we show that CD25-expressing hyperactivated T-cells produce the protease Furin, which facilitates the viral entry of SARS-CoV-2. Collectively, CD4+ T-cells from severe COVID-19 patients are hyperactivated and FOXP3-mediated negative feedback mechanisms are impaired in the lung, which may promote immunopathology. Therefore, our study proposes a new model of T-cell hyperactivation and paralysis that drives immunopathology in severe COVID-19.

The multifaceted Foxp3fgfp allele enhances spontaneous and therapeutic immune surveillance of cancer in mice.

It is well established that therapeutic impairment of Foxp3+ Treg in mice and humans favors immune rejection of solid tumors. Less explored is the impact Foxp3 allelic variants may have on tumor incidence, progression and therapy. In this work, we tested and demonstrate that the Foxp3fgfp reporter allele, found previously to either enhance or reduce Treg function in specific autoimmunity settings, confers increased anti-tumor immunity. Our conclusions stem out of the analysis of three tumor models of different tissue origin, in two murine genetic backgrounds. When compared to wild type animals, mice carrying the Foxp3fgfp allele spontaneously delay, reduce or prevent primary tumor growth, decrease metastasis growth, and potentiate the response to anti-CTLA4 monotherapy. These findings suggest allelic variances at the Foxp3 locus may serve as predictive indicators for personalized therapy and prognostics, and point at possible new therapeutic targets.

Identification of Dmrt2a downstream genes during zebrafish early development using a timely controlled approach.

BACKGROUND: Dmrt2a is a zinc finger like transcription factor with several roles during zebrafish early development: left-right asymmetry, synchronisation of the somite clock genes and fast muscle differentiation. Despite the described functions, Dmrt2a mechanism of action is unknown. Therefore, with this work, we propose to identify Dmrt2a downstream genes during zebrafish early development. RESULTS: We generated and validated a heat-shock inducible transgenic line, to timely control dmrt2a overexpression, and dmrt2a mutant lines. We characterised dmrt2a overexpression phenotype and verified that it was very similar to the one described after knockdown of this gene, with left-right asymmetry defects and desynchronisation of somite clock genes. Additionally, we identified a new phenotype of somite border malformation. We generated several dmrt2a mutant lines, but we only detected a weak to negligible phenotype. As dmrt2a has a paralog gene, dmrt2b, with similar functions and expression pattern, we evaluated the possibility of redundancy. We found that dmrt2b does not seem to compensate the lack of dmrt2a. Furthermore, we took advantage of one of our mutant lines to confirm dmrt2a morpholino specificity, which was previously shown to be a robust knockdown tool in two independent studies. Using the described genetic tools to perform and validate a microarray, we were able to identify six genes downstream of Dmrt2a: foxj1b, pxdc1b, cxcl12b, etv2, foxc1b and cyp1a. CONCLUSIONS: In this work, we generated and validated several genetic tools for dmrt2a and identified six genes downstream of this transcription factor. The identified genes will be crucial to the future understanding of Dmrt2a mechanism of action in zebrafish.