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.

Adoptive Transfer and Bone Marrow Chimera Models to Analyze Treg Function and Differentiation.

Cellular adoptive transfer and mixed bone marrow chimera are cornerstone experimental tools for immuno-biology. Here we describe protocols for adoptive transfer and bone marrow chimera to address the effect of a specific mutation on T regulatory cell (Treg) function and differentiation, respectively. Treg function can be quantitatively measured by analyzing the expansion of conventional CD4 T cells and their differentiation into helper cells. The quantitative measure of Treg differentiation is addressed by analyzing the number and phenotype of Foxp3-expressing cells. The use of congenic markers is instrumental for these approaches.

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.

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.

Recent thymic emigrants are the preferential precursors of regulatory T cells differentiated in the periphery.

Most Forkhead box P3(+) (Foxp3(+)) CD4 regulatory T cell (Treg) precursors are newly formed thymocytes that acquire Foxp3 expression on antigen encounter in the thymus. Differentiation of Treg, however, can also occur in the periphery. What limits this second layer of self- and nonself-reactive Treg production in physiological conditions remains to be understood. In this work, we tested the hypothesis that, similarly to thymic Treg, the precursors of peripheral Treg are immature T cells. We show that CD4(+)CD8(-)Foxp3(-) thymocytes and recent thymic emigrants (RTEs), contrarily to peripheral naïve mature cells, efficiently differentiate into Treg on transfer into lymphopenic mice. By varying donor and recipient mice and conducting ex vivo assays, we document that the preferential conversion of newly formed T cells does not require intrathymic preactivation, is cell-intrinsic, and correlates with low and high sensitivity to natural inhibitors and inducers of Foxp3 expression, such as IL-6, T-cell receptor triggering, and TGF-ß. Finally, ex vivo analysis of human thymocytes and peripheral blood T cells revealed that human RTE and newly developed T cells share an increased potential to acquire a FOXP3(bright)CD25(high) Treg phenotype. Our findings indicating that RTEs are the precursors of Tregs differentiated in the periphery should guide the design of Treg-based therapies.

Cutting edge: Intrathymic differentiation of adaptive Foxp3+ regulatory T cells upon peripheral proinflammatory immunization.

Thymocytes differentiate into CD4(+) Foxp3(+) regulatory T cells (T(R)) upon interaction between their TCR and peptide-MHC II complexes locally expressed in the thymus. Conversion of naive CD4(+) T cells into T(R) can additionally take place in the periphery under noninflammatory conditions of Ag encounter. In this study, making use of TCR transgenic models naturally devoid of Foxp3(+) cells, we report de novo generation of T(R) upon a single footpad injection of Ag mixed with a classic proinflammatory adjuvant. Abrupt T(R) differentiation upon immunization occurred intrathymically and was essential for robust tolerance induction in a mouse model of spontaneous encephalomyelitis. This phenomenon could be attributed to a specific feature of thymocytes, which, in contrast to mature peripheral CD4(+) T cells, were insensitive to the inhibitory effects of IL-6 on the induction of Foxp3 expression. Our findings uncover a pathway for T(R) generation with major implications for immunity and tolerance induction.

Defective induction of CTLA-4 in the NOD mouse is controlled by the NOD allele of Idd3/IL-2 and a novel locus (Ctex) telomeric on chromosome 1.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), or CD152, is a negative regulator of T-cell activation and has been shown to be associated with autoimmune diseases. Previous work has demonstrated a defect in the expression of this molecule in nonobese diabetic (NOD) mice upon anti-CD3 stimulation in vitro. Using a genetic approach we here demonstrate that a novel locus (Ctex) telomeric on chromosome 1 together with the Idd3 (Il-2) gene confers optimal CTLA-4 expression upon CD3 activation of T-cells. Based on these data, we provide a model for how gene interaction between Idd3 (IL-2), Ctex, and Idd5.1 (Ctla-4) could confer susceptibility to autoimmune diabetes in the NOD mouse. Additionally, we showed that the Ctex and the Idd3 regions do not influence inducible T-cell costimulator (ICOS) protein expression in NOD mice. Instead, as previously shown, higher ICOS levels in NOD mice appear to be controlled by gene(s) in the Idd5.1 region, possibly a polymorphism in the Icos gene itself.

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11.

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 x NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.