Michael A. Farrar

IL-2 Receptor β-Dependent STAT5 Activation Is Required for the Development of Foxp3+ Regulatory T Cells

IL-2−/− mice develop autoimmunity despite having relatively normal numbers of regulatory T cells (Tregs). In contrast, we demonstrate that IL-2−/− × IL-15−/− and IL-2Rβ−/− mice have a significant decrease in Treg numbers. Ectopic expression of foxp3 in a subset of CD4+ T cells rescued Treg development and prevented autoimmunity in IL-2Rβ−/− mice, suggesting that IL-2Rβ-dependent signals regulate foxp3 expression in Tregs. Subsequent analysis of IL-2Rβ-dependent signal transduction pathways established that the transcription factor STAT5 is necessary and sufficient for Treg development. Specifically, T cell-specific deletion of STAT5 prevented Treg development; conversely, reconstitution of IL-2Rβ−/− mice with bone marrow cells expressing an IL-2Rβ mutant that exclusively activates STAT5 restored Treg development. Finally, STAT5 binds to the promoter of the foxp3 gene suggesting that IL-2Rβ-dependent STAT5 activation promotes Treg differentiation by regulating expression of foxp3.

Linked T Cell Receptor and Cytokine Signaling Govern the Development of the Regulatory T cell Repertoire

Appropriate development of regulatory T (Treg) cells is necessary to prevent autoimmunity. Neonatal mice, unlike adults, lack factors required for Treg cell development. It is unclear what these missing factors are. However, signals emanating from the T cell receptor (TCR), the costimulatory receptor CD28, and the family of gammac-dependent cytokine receptors are required for Treg cell development. Herein we demonstrate that expression of a constitutively active Stat5b transgene (Stat5b-CA) allowed for Treg cell development in neonatal mice and restored Treg cell numbers in Cd28(-/-) mice. Sequence analysis of TCR genes in Stat5b-CA Treg cells indicated that ectopic STAT5 activation resulted in a TCR repertoire that more closely resembled that of naive T cells. Using MHCII tetramers to identify antigen-specific T cells, we showed that STAT5 signals diverted thymocytes normally destined to become naive T cells into the Treg cell lineage. Our data support a two-step model of Treg cell differentiation in which TCR and CD28 signals induce cytokine responsiveness and STAT5-inducing cytokines then complete the program of Treg cell differentiation.

Distinct IL-2 Receptor Signaling Pattern in CD4+CD25 + Regulatory T Cells

Despite expression of the high-affinity IL-2R, CD4+CD25+ regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4+CD25+ T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4+CD25+ Tregs to IL-2. IL-2R stimulation results in a G1 cell cycle arrest, cellular enlargement and increased cellular survival of CD4+CD25+ T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4+CD25+ T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.

In Vivo Survival and Homeostatic Proliferation of Natural Killer Cells

While the specificity and development of natural killer (NK) cells have been intensely studied, little is known about homeostasis of the mature NK population. Here we show that mouse NK cells undergo homeostatic proliferation when transferred into NK-deficient Rag−/− γC−/− hosts. Normal NK functional activity is maintained during this process, although there are some changes in NK phenotype. Using cell sorting, we demonstrate that mature (Mac-1hi) NK cells undergo homeostatic proliferation in an NK-deficient environment, yet immature (Mac-1lo) NK cells also proliferate in such hosts. We find that mature NK cells survive but do not proliferate in hosts which possess an endogenous NK pool. However, we go on to show that mature NK survival is critically dependent on interleukin (IL)-15. Surprisingly, NK survival is also compromised after transfer of cells into IL-15Rα−/− mice, implying that IL-15 responsiveness by bystander cells is critical for NK maintenance. These data imply that, similar to T cells, homeostasis of the NK pool is much more dynamic than previously appreciated and this may be relevant to manipulation of NK cells for therapeutic purposes.

IL-2, -7, and -15, but Not Thymic Stromal Lymphopoeitin, Redundantly Govern CD4+Foxp3+ Regulatory T Cell Development

Common γ chain (γc)-receptor dependent cytokines are required for regulatory T cell (Treg) development as γc−/− mice lack Tregs. However, it is unclear which γc-dependent cytokines are involved in this process. Furthermore, thymic stromal lymphopoietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing CD4+Foxp3+ Tregs in the thymus express the IL-2Rβ, IL-4Rα, IL-7Rα, IL-15Rα, and IL-21Rα chains, but not the IL9Rα or TSLPRα chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in CD4+Foxp3+ Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were capable of inducing the conversion of CD4+CD25+Foxp3− thymic Treg progenitors into CD4+Foxp3+ mature Tregs in vitro. To examine this issue in more detail, we generated IL-2Rβ−/− × IL-7Rα−/− and IL-2Rβ−/− × IL-4Rα−/− mice. We found that IL-2Rβ−/− × IL-7Rα−/− mice were devoid of Tregs thereby recapitulating the phenotype observed in γc−/− mice; in contrast, the phenotype observed in IL-2Rβ−/− × IL-4Rα−/− mice was comparable to that seen in IL-2Rβ−/− mice. Finally, we observed that Tregs from both IL-2−/− and IL-2Rβ−/− mice show elevated expression of IL-7Rα and IL-15Rα chains. Addition of IL-2 to Tregs from IL-2−/− mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the predominant role in Treg development, but that in its absence the IL-7Rα and IL-15Rα chains are up-regulated and allow for IL-7 and IL-15 to partially compensate for loss of IL-2.

Distinct Effects of STAT5 Activation on CD4+ and CD8+ T Cell Homeostasis: Development of CD4+CD25+ Regulatory T Cells versus CD8+ Memory T Cells

Using transgenic mice that express a constitutively active version of STAT5b, we demonstrate that STAT5 plays a key role in governing B cell development and T cell homeostasis. STAT5 activation leads to a 10-fold increase in pro-B, but not pro-T, cells. Conversely, STAT5 signaling promotes the expansion of mature αβ T cells (6-fold increase) and γδ and NK T cells (3- to 4-fold increase), but not of mature B cells. In addition, STAT5 activation has dramatically divergent effects on CD8+ vs CD4+ T cells, leading to the selective expansion of CD8+ memory-like T cells and CD4+CD25+ regulatory T cells. These results establish that activation of STAT5 is the primary mechanism underlying both IL-7/IL-15-dependent homeostatic proliferation of naive and memory CD8+ T cells and IL-2-dependent development of CD4+CD25+ regulatory T cells.

Immune tolerance. Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4⁺ T cells.

Innate lymphoid cells keep gut T cells in check Trillions of bacteria inhabit our guts. So do many types of immune cells, including T cells, which might be expected to attack these bacteria. How, then, do our bodies manage to keep the peace? Working in mice, Hepworth et al. report one such mechanism. A population of immune cells, called innate lymphoid cells, directly killed CD4+ T cells that react to commensal gut microbes. Some of the specifics of this process parallel how the immune system keeps developing self-reactive T cells in check in the thymus. Furthermore, this peacekeeping process may be disrupted in children with inflammatory bowel disease. Science, this issue p. 1031 Innate lymphoid cells delete commensal bacteria–specific CD4+ T cells from the intestine in mice. Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. Although selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria–specific T cells remain poorly understood. Here, we demonstrate that group 3 innate lymphoid cell (ILC3)–intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria–specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria–specific CD4+ T cells in the intestine and suggest that this process is dysregulated in human IBD.

RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1–RAG-2 recombinase and was restored in Rag1−/− developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

STAT5 Activation Underlies IL7 Receptor-Dependent B Cell Development

Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R−/− background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R−/− mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-xL expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R−/− mice as determined by 1) the restoration of VH Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.

Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells

Regulatory T (Treg) cells express tumor necrosis factor receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors highly express the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors correlates directly with T cell receptor (TCR) signal strength, and requires CD28 and the kinase TAK1. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation by augmenting IL-2R/STAT5 responsiveness. GITR-ligand costimulation elicited a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on Treg progenitors translates strong TCR signals into molecular parameters that specifically promote Treg differentiation and shape the Treg repertoire.Regulatory T cells (Treg cells) express members of the tumor-necrosis factor (TNF) receptor superfamily (TNFRSF), but the role of those receptors in the thymic development of Treg cells is undefined. We found here that Treg cell progenitors had high expression of the TNFRSF members GITR, OX40 and TNFR2. Expression of those receptors correlated directly with the signal strength of the T cell antigen receptor (TCR) and required the coreceptor CD28 and the kinase TAK1. The neutralization of ligands that are members of the TNF superfamily (TNFSF) diminished the development of Treg cells. Conversely, TNFRSF agonists enhanced the differentiation of Treg cell progenitors by augmenting responsiveness of the interleukin 2 receptor (IL-2R) and transcription factor STAT5. Costimulation with the ligand of GITR elicited dose-dependent enrichment for cells of lower TCR affinity in the Treg cell repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated the development of Treg cells. Thus, expression of members of the TNFRSF on Treg cell progenitors translated strong TCR signals into molecular parameters that specifically promoted the development of Treg cells and shaped the Treg cell repertoire.