Nan-Shih Liao

Reduced Expression of Bcl-2 in CD8+ T Cells Deficient in the IL-15 Receptor α-Chain

Mice that lack IL-15 or the IL-15R α-chain (IL-15Rα) are deficient in peripheral CD8+, but not in CD4+, T cells. This CD8+ T cell-specific deficiency has now been investigated further by characterization of a new strain of IL-15Rα−/− mice. The adult mutant mice exhibited a specific reduction in the percentage of CD8-single positive TCRhigh thymocytes. The expression of Bcl-2 was reduced in both CD8+ thymocytes and naive T cells of the mutant animals, and the susceptibility of these cells to death was increased. Memory CD8+ cells were profoundly deficient in IL-15Rα−/−mice, and the residual memory-like CD8+ cells contained a high percentage of dead cells and failed to up-regulate Bcl-2 expression compared with naive CD8+ cells. Moreover, exogenous IL-15 both up-regulated the level of Bcl-2 in and reduced the death rate of wild-type and mutant CD8+ T cells activated in vitro. These results indicate that IL-15 and IL-15Rα regulate the expression of Bcl-2 in CD8+ T cells at all developmental stages. The reduced Bcl-2 content in CD8+ cells might result in survival defect and contribute to the reduction of CD8+ cells in IL-15Rα−/−mice.

Different NK Cell Developmental Events Require Different Levels of IL-15 Trans-Presentation

NK cell development requires IL-15, which is “trans-presented” to IL-15Rβγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)–derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.

IL-15 Does Not Affect IEL Development in the Thymus but Regulates Homeostasis of Putative Precursors and Mature CD8αα+ IELs in the Intestine

Mice devoid of the IL-15 system lose over 90% of CD8αα+ TCRαβ and TCRγδ intestinal intraepithelial lymphocytes (iIELs). Previous work revealed that IL-15Rα and IL-15 expressed by parenchymal cells, but not by bone marrow-derived cells, are required for normal CD8αα+ iIEL homeostasis. However, it remains unclear when and how the IL-15 system affects CD8αα+ iIELs through their development. This study found that IL-15Rα is dispensable for the thymic stage of CD8αα+ TCRαβ and TCRγδ iIEL development but is required for the maintenance and/or differentiation of the putative lineage marker negative precursors in the intestinal epithelium, especially for the most mature CD8 single positive subset. Moreover, the IL-15 system directly supports the survival of mature CD8αα+ iIEL in vivo. Taken together, this study suggests that regulation of CD8αα+ iIEL homeostasis by the IL-15 system does not occur in the thymus but involves mature cells and putative precursors in the intestine.

IL-15 modulates the balance between Bcl-2 and Bim via a Jak3/1-PI3K-Akt-ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα+ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα+ iIELs remains elusive. Although Bcl‐2 level in CD8αα+ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα+ γδ iIELs in Il15−/− mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser65. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα+ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα+ iIELs promoted their survival in Il15ra−/− mice. Taken together, IL‐15 promotes CD8αα+ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.

Reduced 2,4‐dinitro‐1‐fluorobenzene‐induced contact hypersensitivity response in IL‐15 receptor α‐deficient mice correlates with diminished CCL5/RANTES and CXCL10/IP‐10 expression

Using a model of 2,4‐dinitro‐1‐fluorobenzene‐induced contact hypersensitivity (CHS) we found that, as compared with wild‐type mice, IL‐15 receptor α chain (IL‐15Rα)‐deficient mice showed significantly less ear swelling. This decreased response was associated with diminished expression of CCL5/RANTES and CXCL10/IP‐10, chemokines critical for effector cell recruitment, in the inflamed tissue. We determined that both the number of CD8+ T cells infiltrating the affected skin and the production of CCL5/RANTES by antigen‐stimulated CD8+ T cells were decreased in IL‐15Rα–/– mice. The lower levels of CXCL10/IP‐10 suggested that the IL‐15Rα–/– mice had reduced production of IFN‐γ, the primary inducer of CXCL10/IP‐10, which was in fact the case. However, by contrast with CCL5/RANTES, the diminished levels of IFN‐γ were likely due to the decreased number of skin‐infiltrating CD8+ T cells, since IFN‐γ production by antigen‐stimulated CD8+ T cells was comparable between wild‐type and IL‐15Rα–/– mice. Our data suggest a positive, pro‐inflammatory feedback loop involving CCL5/RANTES, IFN‐γ and CXCL10/IP‐10 that underlies the CHS reaction and that is disrupted, likely primarily by a defect in CCL5/RANTES production, in mice lacking IL‐15Rα, resulting in impaired leukocyte recruitment and inflammation. Moreover, it is particularly noteworthy that the defect in CCL5/RANTES expression in CD8+ T cells is intrinsic to the absence of IL‐15Rα, indicating that IL‐15Rα is critical for CCL5/RANTES expression in CD8+ T cells.

IL-15Rα of Radiation-Resistant Cells Is Necessary and Sufficient for Thymic Invariant NKT Cell Survival and Functional Maturation

The development of invariant NKT (iNKT) cells depends on the thymus. After positive selection by CD4+CD8+CD1d+ cortical thymocytes, iNKT cells proceed from CD44lowNK1.1− (stage 1) to CD44highNK1.1− (stage 2), and then to CD44highNK1.1+ (stage 3) cells. The programming of cytokine production occurs along the three differentiation stages, whereas the acquisition of NK receptors occurs at stage 3. Stage 3 thymic iNKT cells are specifically reduced in Il15ra−/− mice. The mechanism underlying this homeostatic deficiency and whether the IL-15 system affects other thymic iNKT cell developmental events remain elusive. In this study, we demonstrate that increased cell death contributed to the reduction of stage 3 cells in Il15ra−/− mice, as knockout of Bim restored this population. IL-15–dependent upregulation of Bcl-2 in stage 3 cells affected cell survival, as overexpression of hBcl-2 partially restored stage 3 cells in Il15ra−/− mice. Moreover, thymic iNKT cells in Il15ra−/− mice were impaired in functional maturation, including the acquisition of Ly49 and NKG2 receptors and the programming of cytokine production. Finally, IL-15Rα expressed by radiation-resistant cells is necessary and sufficient to support the survival as well as the examined maturation events of thymic iNKT cells.

Adipocyte IL-15 Regulates Local and Systemic NK Cell Development

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15−/− mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15−/− mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient’s spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.

Skeletal muscle interleukin 15 promotes CD8(+) T-cell function and autoimmune myositis.

BackgroundInterleukin 15 (IL-15) is thought to be abundant in the skeletal muscle under steady state conditions based on RNA expression; however, the IL-15 RNA level may not reflect the protein level due to post-transcriptional regulation. Although exogenous protein treatment and overexpression studies indicated IL-15 functions in the skeletal muscle, how the skeletal muscle cell uses IL-15 remains unclear. In myositis patients, IL-15 protein is up-regulated in the skeletal muscle. Given the supporting role of IL-15 in CD8+ T-cell survival and activation and the pathogenic role of cytotoxic CD8+ T cells in polymyositis and inclusion-body myositis, we hypothesize that IL-15 produced by the inflamed skeletal muscle promotes myositis via CD8+ T cells.MethodsExpression of IL-15 and IL-15 receptors at the protein level by skeletal muscle cells were examined under steady state and cytokine stimulation conditions. The functions of IL-15 in the skeletal muscle were investigated using Il15 knockout (Il15−/−) mice. The immune regulatory role of skeletal muscle IL-15 was determined by co-culturing cytokine-stimulated muscle cells and memory-like CD8+ T cells in vitro and by inducing autoimmune myositis in skeletal-muscle-specific Il15−/− mice.ResultsWe found that the IL-15 protein was not expressed by skeletal muscle cells under steady state condition but induced by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) stimulation and expressed as IL-15/IL-15 receptor alpha (IL-15Rα) complex. Skeletal muscle cells expressed a scanty amount of IL-15 receptor beta (IL-15Rβ) under either conditions and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ. On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8+ T cells. Genetic ablation of Il15 in skeletal muscle cells greatly ameliorated autoimmune myositis in mice.ConclusionsThese findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

Thymic epithelial β-catenin is required for adult thymic homeostasis and function

The role of β‐catenin in thymocyte development has been extensively studied, however, the function of β‐catenin in thymic epithelial cells (TECs) remains largely unclear. Here, we demonstrate a requirement for β‐catenin in keratin 5 (K5)‐expressing TECs, which comprise the majority of medullary TECs (mTECs) and a progenitor subset for cortical TECs (cTECs) in the young adult thymus. We found that conditionally ablated β‐catenin in K5+‐TECs and their progeny cells resulted in thymic atrophy. The composition of TECs was also aberrantly affected. Percentages of K5hiK8+‐TECs, K5+K8–‐TECs and UEA1+‐mTECs were significantly decreased and the percentage of K5loK8+‐TECs and Ly51+‐cTECs were increased in β‐catenin‐deficient thymi compared with that in the control thymi. We also observed that β‐catenin‐deficient TEC lineage could give rise to K8+‐cTECs more efficiently than wild‐type TECs using lineage‐tracing approach. Importantly, the expression levels of several transcription factors (p63, FoxN1 and Aire), which are essential for TEC differentiation, were altered in β‐catenin‐deficient thymi. Under the aberrant differentiation of TECs, development of all thymocytes in β‐catenin‐deficient thymi was impaired. Interleukin‐7 (IL‐7) and chemokines (Ccl19, Ccl25 and Cxcl12) levels were also downregulated in the thymic stromal cells in the mutants. Finally, introducing a BCL2 transgene in lymphoid lineages, which has been shown to rescue IL‐7‐deficient thymopoiesis, partially rescued the thymic atrophy and thymocyte development defects caused by induced ablation of β‐catenin in K5+‐TECs. Collectively, these findings suggest that β‐catenin is required for the differentiation of TECs, thereby contributing to thymocyte development in the postnatal thymus.

Promoter Knock-In Mutations Reveal a Role of Mcl-1 in Thymocyte-Positive Selection and Tissue or Cell Lineage-Specific Regulation of Mcl-1 Expression

We previously demonstrated that IL-3 stimulates transcription of the antiapoptotic gene mcl-1 via two promoter elements designated as the SIE and CRE-2 sites. To further study the functional role of these two DNA elements, mutant mice with targeted mutations of both SIE and CRE-2 sites (SC mutants) were generated. Homozygous SC mutants manifested a markedly reduced level of Mcl-1 in thymus but not in other major organs such as spleen, liver, lung, or heart. Reduced expression of Mcl-1 in SC mutant thymus resulted in attenuated positive selection of double-positive thymocytes into both CD4 and CD8 lineages, a result likely due to reduced survival of SC mutant double-positive thymocytes that were supposed to be positively selected. In contrast, in the peripheral lymphoid organs, only CD8+ but not CD4+ T cells were significantly reduced in homozygous SC mutant mice, a result consistent with a more dramatic decrease both of Mcl-1 expression and cell viability in mutant CD8+ compared with mutant CD4+ T cells. Impaired T cell development and peripheral CD8+ lymphopenia in homozygous SC mutant mice were both cell autonomous and could be rescued by enforced expression of human Mcl-1. Together, the promoter-knock-in mouse model generated in this study not only revealed a role of Mcl-1 in thymocyte-positive selection, but also uncovered that Mcl-1 expression is regulated in a tissue or cell lineage-specific manner.