BackgroundThe Fas pathway is a major regulator of T cell homeostasis, however, the T cell population that is controlled by the Fas pathway in vivo is poorly defined. Although CD4 and CD8 single positive (SP) T cells are the two major T cell subsets in the periphery of wild type mice, the repertoire of mice bearing loss-of-function mutation in either Fas (lpr mice) or Fas ligand (gld mice) is predominated by CD4−CD8− double negative αβ T cells that also express B220 and generally referred to as B220+DN T cells. Despite extensive analysis, the basis of B220+DN T cell lymphoproliferation remains poorly understood. In this study we re-examined the issue of why T cell lymphoproliferation caused by gld mutation is predominated by B220+DN T cells.Methodology and Principal FindingsWe combined the following approaches to study this question: Gene transcript profiling, BrdU labeling, and apoptosis assays. Our results show that B220+DN T cells are proliferating and dying at exceptionally high rates than SP T cells in the steady state. The high proliferation rate is restricted to B220+DN T cells found in the gut epithelium whereas the high apoptosis rate occurred both in the gut epithelium and periphery. However, only in the periphery, apoptosis of B220+DN T cell is Fas-dependent. When the Fas pathway is genetically impaired, apoptosis of peripheral B220+DN T cells was reduced to a baseline level similar to that of SP T cells. Under these conditions of normalized apoptosis, B220+DN T cells progressively accumulate in the periphery, eventually resulting in B220+DN T cell lymphoproliferation.Conclusions/SignificanceThe Fas pathway plays a critical role in regulating the tissue distribution of DN T cells through targeting and elimination of DN T cells from the periphery in the steady state. The results provide new insight into pathogenesis of DN T cell lymphoproliferation.

The Fas pathway and regulatory T (T(R)) cells play intertwining roles in controlling T cell tolerance through deletion and suppression of autoreactive T cells. Impairment of either mechanism causes severe T cell lymphoproliferation albeit with opposing outcomes. T cell lymphoproliferation induced by defective Fas pathway does not cause overt lymphocytic infiltration but rather prevents an important set of T cell-mediated autoimmune diseases. In contrast, deficiency in T(R) cell causes fulminant autoimmunity in very early life and fatal lymphocytic infiltration. These observations suggest existence of unidirectional fail/safe mechanism that compensate for defects in the Fas pathway but not in regulatory cells. To gain insights into how animals compensate for defects in the Fas system, we analyzed the impact of generalized lymphoproliferative disease (gld) mutation on survival, function and transcription profile of CD25+CD4+ T(R) cells. Our results show that all CD4 T cells expanded in gld mice. However, CD25+CD4+ T(R) cells are disproportionately increased in the pool of CD4 T cells perhaps due to their unique apoptosis phenotype. Freshly isolated CD25+CD4+ T(R) cells, unlike CD25-CD4+ T cells, are highly sensitive to FasL-induced apoptosis in the steady state. CD25+CD4+ T(R) cells that accumulate in gld mice express similar level of Foxp3, and have suppression potency and T(R) gene expression profile as wild-type CD25+CD4+ T(R) cells. Furthermore, the transcription profile of gld CD25+CD4+ T(R) cells is characterized by differential expression of genes involved in cell survival, metabolism and innate immune responses. These results provide a strong cellular and molecular basis for understanding why impaired Fas pathway prevents an important subset of T cell-mediated autoimmune diseases.

Summary Invariant natural killer T (iNKT) cells recognize glycolipids as antigens and diversify into NKT1 (IFN-γ), NKT2 (IL-4), and NKT17 (IL-17) functional subsets while developing in the thymus. Mechanisms that govern the balance between these functional subsets are poorly understood due partly to the lack of distinguishing surface markers. Here we identified the heparan sulfate proteoglycan syndecan-1 (sdc1) as a specific marker of naïve thymic NKT17 cells and that sdc1 deficiency significantly increased thymic NKT17 cells at the expense of NKT1 cells, leading to impaired iNKT cell-derived IFN-γ, both in vitro and in vivo. Using surface expression of sdc1 to identify NKT17 cells, we confirmed differential tissue localization and interstrain variability of NKT17 cells and uncovered that NKT17 cells expressed high TCRβ, preferentially use Vβ8, and display high sensitivity to ɑ-GalCer than to CD3/CD28 stimulation. These findings provide a novel non-invasive simple method for identification and viable sorting of naïve NKT17 cells from unmanipulated mice and suggest that sdc1 expression negatively regulates homeostasis iNKT cells. In addition, they lay the groundwork for investigating the mechanisms by which sdc1 regulates NKT17 cells.