Regulatory T (Treg) cells play a central role in maintaining immune homeostasis. However, little is known about the stability of Treg cells in vivo. In this study, we demonstrate that a significant percentage of cells exhibited transient or unstable Foxp3 expression. These exFoxp3+ T cells express an activated-memory T cell phenotype, and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers increased in inflamed tissues under autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid-onset of diabetes. Finally, T cell receptor repertoire analyses suggested that exFoxp3 cells develop from both natural and adaptive Treg cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.

A new regulatory T (T reg) cell–specific, FoxP3-GFP-hCre bacterial artificial chromosome transgenic mouse was crossed to a conditional Dicer knockout (KO) mouse strain to analyze the role of microRNAs (miRNAs) in the development and function of T reg cells. Although thymic T reg cells developed normally in this setting, the cells showed evidence of altered differentiation and dysfunction in the periphery. Dicer-deficient T reg lineage cells failed to remain stable, as a subset of cells down-regulated the T reg cell–specific transcription factor FoxP3, whereas the majority expressed altered levels of multiple genes and proteins (including Neuropilin 1, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4) associated with the T reg cell fingerprint. In fact, a significant percentage of the T reg lineage cells took on a T helper cell memory phenotype including increased levels of CD127, interleukin 4, and interferon γ. Importantly, Dicer-deficient T reg cells lost suppression activity in vivo; the mice rapidly developed fatal systemic autoimmune disease resembling the FoxP3 KO phenotype. These results support a central role for miRNAs in maintaining the stability of differentiated T reg cell function in vivo and homeostasis of the adaptive immune system.

Aire regulates medullary epithelial cell production of XCL1, a chemoattractant for XCR1-expressing thymic DCs whose presence in the medulla contributes to the generation of T reg cells.

SummaryRegulatory T (Treg) cells play an essential role in maintaining immunological tolerance. The discovery of FoxP3 as a key Treg transcription factor combined with recent advances in the development of functional reporter mice have enabled new insights into Treg biology and revealed unexpected features of this lineage. In this review, we address the stability of this population, focusing on studies that suggest that Tregs can down-regulate FoxP3, lose regulatory activity and, under some conditions, become memory T cells capable of recognizing self-antigens and expressing effector cell activities including the production of IL-17 and IFNγ. The presence of these "exTregs" in multiple inflammatory settings suggests a potential role for these cells in a variety of disease settings ranging from autoimmunity to cancer and infectious disease.

T follicular helper (TFH) cells are the prototypic helper T cell subset specialized to enable B cells to form germinal centers and produce high-affinity antibodies. We found that miRNA expression by T cells was essential for TFH cell differentiation. More specifically, we show that after protein immunization the microRNA cluster miR-17~92 was critical for robust TFH cell differentiation and function in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17~92 restrained the expression of TFH subset-inappropriate genes, including the direct target RAR-related orphan receptor alpha (Rora). Genetically removing one Rora allele partially rescued the inappropriate gene signature in miR-17~92-deficient TFH cells. Our results identify the miR-17~92 cluster as a critical regulator of T cell-dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene expression program.

The systemic administration of keratinocyte growth factor (KGF) enhances T-cell lymphopoiesis in normal mice and mice that received a bone marrow transplant. KGF exerts protection to thymic stromal cells from cytoablative conditioning and graft-versus-host disease-induced injury. However, little is known regarding KGF's molecular and cellular mechanisms of action on thymic stromal cells. IntroductionDecreased T-cell cellularity and a skewed TCR repertoire are hallmarks of an immune deficiency commonly observed in old age, as a consequence of general infectious diseases and aggressive lymphocyte-depleting therapies for diverse malignancies. [1][2][3][4] The regeneration of a phenotypically and functionally normal T-cell compartment is curtailed for an extended period of time in patients receiving a hematopoietic stem cell transplant (HSCT). [5][6][7] This lack in T-cell reconstitution is associated with opportunistic infections, the reactivation of latent viral and parasitic infections, chronic inflammation, and autoimmunity. 3,4 Following cytoablative therapy, the recovery of the T-cell compartment relies on 2 independent pathways, that is, the expansion of peripheral T cells and, alternatively, the de novo production of T cells in the thymus. 1,2,7-10 The latter assures the generation of a population of naive T cells expressing a diverse repertoire of TCR specificities. 5,7,8,10,11 The extent of thymusdependent T-cell reconstitution correlates directly with thymic size following immune ablation and hematopoietic stem cell (HSC)-derived reconstitution 7,12 but is inversely related to age and transplant-related toxicities such as graft-versus-host disease (GVHD). 10,[13][14][15][16][17] The generation of new T cells of donor origin depends on the migration of hematopoietic precursors to the thymus. Normal thymic T-cell development is in turn contingent on the regular maintenance of the stromal microenvironment. However, age-related thymic involution 18 and injury from radiation, 19 GVHD, 20 chemotherapy, 12,21 or infection 3,4,12,[18][19][20][21][22][23] preclude normal thymopoiesis to occur as they directly affect thymic epithelial cells (TECs). There has been considerable interest in identifying strategies to prevent TEC injury. Recently, robust T-cell lymphopoiesis has been maintained in myeloablated HSCT recipients by pretransplantation administration of different factors such as 24,25 androgen antagonists, 26 and fibroblast growth factor 7 (Fgf7; aka, keratinocyte growth factor [KGF]). 20,27-29 KGF belongs to the family of the structurally related Fgfs and is a potent epithelial cell mitogen. 27,30 KGF is expressed under physiological conditions within the thymus both by mesenchymal cells and by T cells at specific developmental stages. To exert its biologic activity, KGF activates the IIIb variant of the FgfR2 receptor (FgfR2IIIb), which is expressed within the thymus exclusively on TECs. 31 Experiments using mice deficient for FgfR2IIIb or the removal of mesenchyme from normal embryos revealed the importa...

Summary The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.

SUMMARY MicroRNAs (miRNAs) are important regulators of cell fate decisions in immune responses. They act by coordinate repression of multiple target genes, a property that we exploited to uncover regulatory networks that govern T helper-2 (Th2) cells. A functional screen of individual miRNAs in primary T cells uncovered multiple miRNAs that inhibited Th2 cell differentiation. Among these were miR-24 and miR-27, miRNAs coexpressed from two genomic clusters, which each functioned independently to limit interleukin-4 (IL-4) production. Mice lacking both clusters in T cells displayed increased Th2 cell responses and tissue pathology in a mouse model of asthma. Gene expression and pathway analyses placed miR-27 upstream of genes known to regulate Th2 cells. They also identified targets not previously associated with Th2 cell biology which regulated IL-4 production in unbiased functional testing. Thus, elucidating the biological function and target repertoire of miR-24 and miR-27 reveals regulators of Th2 cell biology.