A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells.

Interleukin-2 receptor (IL-2R) signaling is essential for T regulatory (Treg) cell development and homeostasis. Here, we show that expression of IL-2Rbeta chains that lack tyrosine residues important for the association of the adaptor Shc and the transcription factor STAT5 in IL-2Rbeta-deficient mice resulted in production of a normal proportion of natural Treg cells that suppressed severe autoimmunity related with deficiency in IL-2 or IL-2R. These mutant IL-2Rbeta chains supported suboptimal and transient STAT5 activation that upregulate the transcription factor Foxp3 to normal amounts in natural, but not induced, Treg cells. Nevertheless, gene expression profiling revealed many targets in peripheral natural Treg cells that were IL-2 dependent and a substantial overlap between the Treg cell IL-2-dependent gene program and the Treg cell transcriptional signature. Collectively, these findings demonstrate that a critical, and perhaps minor, subset of IL-2-dependent targets is indexed to a low IL-2R signaling threshold and that a substantial proportion of the Treg cell gene program is regulated by IL-2.

IL-2R signaling is essential for functional maturation of regulatory T cells during thymic development.

CD4(+) Foxp3(+) regulatory T cells (Tregs) are an independent cell lineage, and their developmental progression during thymic development depends on IL-2R signaling. However, the role of IL-2R signaling during thymic Treg development remains only partially understood. The current study assessed the contribution of IL-2 to the expansion and functional programming of developing Tregs. In the absence of IL-2Rß signaling, predominantly CD4(+) CD25(-) Foxp3(lo) T cells were found, and these cells exhibited somewhat lower expression of the proliferative marker Ki67. These immature Tregs, which represent products of failed development, were also found in normal mice and were characterized by markedly lower expression of several Treg functional molecules. Therefore, IL-2R is required for the progression, functional programming, and expansion of Tregs during thymic development. An IL-2R-signaling mutant that lowers STAT5 activation readily supported Treg functional programming, but Treg proliferation remained somewhat impaired. The requirement for IL-2 during thymic Treg expansion was best illustrated in mixed chimeras where the Tregs with mutant IL-2Rs were forced to compete with wild-type Tregs during their development. Tregs with impaired IL-2R signaling were more prevalent in the thymus than spleen in these competitive experiments. The general effectiveness of mutant IL-2Rs to support thymic Treg development is partially accounted for by a heightened capacity of thymic Tregs to respond to IL-2. Overall, our data support a model in which limiting IL-2R signaling is amplified by thymic Tregs to readily support their development and functional programming, whereas these same conditions are not sufficient to support peripheral Treg homeostasis.

Efficient xenoengraftment in severe immunodeficient NOD/Shi-scid IL2rγnull mice is attributed to a lack of CD11c+B220+CD122+ cells.

Xenograft animal models using immunodeficient mice have been widely applied in medical research on various human diseases. NOD/Shi-scid-IL2rγ(null) (NOG) mice are known to show an extremely high engraftment rate of xenotransplants compared with conventional immunodeficient mice. This high engraftment rate of xenotransplants in NOG mice was substantially suppressed by the transfer of spleen cells from NOD-scid mice that were devoid of NK cells. These results indicate that cell types other than splenic NK cells present in NOD-scid mice but not in NOG mice may be involved in this suppression. To identify the cell types responsible for this effect, we transferred subpopulations of spleen cells from NOD-scid mice into NOG mice and assessed the levels of human cell engraftment after human PBMC (hPBMC) transplantation. These experiments revealed that CD11c(+)B220(+) plasmacytoid dendritic cells (pDCs) from NOD-scid mice markedly inhibited engraftment of human cells. The CD11c(+)B220(+)CD122(+) cells further fractionated from the pDCs based on the expression of CD122, which is an NK cell marker strongly inhibited during hPBMC engraftment in NOG mice. Moreover, the CD122(+) cells in the pDC fraction were morphologically distinguishable from conventional CD122(+) NK cells and showed a higher rejection efficiency. The current results suggest that CD11c(+)B220(+)CD122(+) cells play an important role in xenograft rejection, and their absence in NOG mice may be critical in supporting the successful engraftment of xenotransplants.

The basis of distinctive IL-2- and IL-15-dependent signaling: weak CD122-dependent signaling favors CD8+ T central-memory cell survival but not T effector-memory cell development.

Recent work suggests that IL-2 and IL-15 induce distinctive levels of signaling through common receptor subunits and that such varied signaling directs the fate of Ag-activated CD8(+) T cells. In this study, we directly examined proximal signaling by IL-2 and IL-15 and CD8(+) T cell primary and memory responses as a consequence of varied CD122-dependent signaling. Initially, IL-2 and IL-15 induced similar p-STAT5 and p-S6 activation, but these activities were only sustained by IL-2. Transient IL-15-dependent signaling is due to limited expression of IL-15Rα. To investigate the outcome of varied CD122 signaling for CD8(+) T cell responses in vivo, OT-I T cells were used from mouse models where CD122 signals were attenuated by mutations within the cytoplasmic tail of CD122 or intrinsic survival function was provided in the absence of CD122 expression by transgenic Bcl-2. In the absence of CD122 signaling, generally normal primary response occurred, but the primed CD8(+) T cells were not maintained. In marked contrast, weak CD122 signaling supported development and survival of T central-memory (T(CM)) but not T effector-memory (T(EM)) cells. Transgenic expression of Bcl-2 in CD122(-/-) CD8(+) T cells also supported the survival and persistence of T(CM) cells but did not rescue T(EM) development. These data indicate that weak CD122 signals readily support T(CM) development largely through providing survival signals. However, stronger signals, independent of Bcl-2, are required for T(EM) development. Our findings are consistent with a model whereby low, intermediate, and high CD122 signaling support T(CM) memory survival, T(EM) programming, and terminal T effector cell differentiation, respectively.

CD4(+) CD25(+) Foxp3(+) T regulatory cells with limited TCR diversity in control of autoimmunity.

The importance of high TCR diversity of T regulatory (Treg) cells for self-tolerance is poorly understood. To address this issue, TCR diversity was measured for Treg cells after transfer into IL-2Rbeta(-/-) mice, which develop lethal autoimmunity because of failed production of Treg cells. In this study, we show that high TCR diversity of pretransferred Treg cells led to selection of therapeutic Treg cells with lower TCR diversity that prevented autoimmunity. Pretransferred Treg cells with lower diversity led to selection of Treg cells through substantial peripheral reshaping with even more restricted TCR diversity that also suppressed autoimmune symptoms. Thus, in a setting of severe breakdown of immune tolerance because of failed production of Treg cells, control of autoimmunity is achieved by only a fraction of the Treg TCR repertoire, but the risk for disease increased. These data support a model in which high Treg TCR diversity is a mechanism to ensure establishing and maintaining self-tolerance.

Intravenous tolerance effectively overcomes enhanced pro-inflammatory responses and experimental autoimmune encephalomyelitis severity in the absence of IL-12 receptor signaling.

Intravenous (i.v.) administration of autoantigen effectively induces Ag-specific tolerance against experimental autoimmune encephalomyelitis (EAE). We and others have shown enhanced EAE severity in mice lacking IL-12 or its receptor, strongly suggesting an immunoregulatory effect of IL-12 signaling. To examine the role of IL-12 responsiveness in autoantigen-induced tolerance in EAE, we administered autoantigen i.v. in two distinct treatment regimes to wildtype and IL-12Rß2(-/-) mice, immunized to develop EAE. Administration at the induction phase suppressed EAE in wildtype and IL-12Rß2(-/-) mice however the effect was somewhat less potent in the absence of IL-12Rß2. Expression of pro-inflammatory cytokines such as IFN-γ, IL-17 and IL-2, was inhibited in wild-type tolerized mice but less so in IL-12Rß2(-/-) mice. I.v. antigen was also effective in suppressing disease in both genotypes when given during the clinical phase of disease with similar CNS inflammation, demyelination and peripheral inflammatory cytokine profiles observed in both genotypes. There was however a mild impact of a lack of IL-12 signaling on Treg induction during tolerance induction compared to WT mice in this treatment regime. These findings show that the enhanced severity of EAE that occurs in the absence of IL-12 signaling can be effectively overcome by i.v. autoantigen, indicating that this therapeutic effect is not primarily mediated by IL-12 and that i.v. tolerance could be a powerful approach in suppressing severe and aggressive MS.