Scot D. Liu

Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages.

Natural killer (NK) cells attack tumor and infected cells, but the receptors and ligands that stimulate them are poorly understood. Here we report the expression cloning of two murine ligands for the lectin-like receptor NKG2D. The two ligands, H-60 and Rae1β, are distant relatives of major histocompatibility complex class I molecules. NKG2D ligands are not expressed by most normal cells but are up-regulated on numerous tumor cells. We show that mouse NKG2D is expressed by NK cells, activated CD8+ T cells and activated macrophages. Expression of either NKG2D ligand by target cells triggers NK cell cytotoxicity and interferon-γ secretion by NK cells, as well as nitric oxide release and tumor necrosis factor α transcription by macrophages. Thus, through their interaction with NKG2D, H-60 and Rae1β are newly identified potent stimulators of innate immunity.

Osteopontin promotes fibrosis in dystrophic mouse muscle by modulating immune cell subsets and intramuscular TGF-β

Duchenne muscular dystrophy (DMD) is an X-linked, degenerative muscle disease that is exacerbated by secondary inflammation. Here, we characterized the immunological milieu of dystrophic muscle in mdx mice, a model of DMD, to identify potential therapeutic targets. We identified a specific subpopulation of cells expressing the Vbeta8.1/8.2 TCR that is predominant among TCR-beta+ T cells. These cells expressed high levels of osteopontin (OPN), a cytokine that promotes immune cell migration and survival. Elevated OPN levels correlated with the dystrophic process, since OPN was substantially elevated in the serum of mdx mice and muscle biopsies after disease onset. Muscle biopsies from individuals with DMD also had elevated OPN levels. To test the role of OPN in mdx muscle, mice lacking both OPN and dystrophin were generated and termed double-mutant mice (DMM mice). Reduced infiltration of NKT-like cells and neutrophils was observed in the muscle of DMM mice, supporting an immunomodulatory role for OPN in mdx muscle. Concomitantly, an increase in CD4+ and FoxP3+ Tregs was also observed in DMM muscle, which also showed reduced levels of TGF-beta, a known fibrosis mediator. These inflammatory changes correlated with increased strength and reduced diaphragm and cardiac fibrosis. These studies suggest that OPN may be a promising therapeutic target for reducing inflammation and fibrosis in individuals with DMD.

Galectin-1 functions as a Th2 cytokine that selectively induces Th1 apoptosis and promotes Th2 function

Galectin‐1 has been implicated in regulating T‐cell survival, function, and Th1/Th2 balance in several mouse models, though the molecular and cellular basis of its immuno‐modulatory activity has not been completely elucidated. Therefore, we examined galectin‐1 expression and activity within differentiated murine Th1 and Th2 subsets. While recombinant galectin‐1 specifically bound to both T‐cell subsets, Th1 and Th2 T cells expressed distinct combinations of galectin‐1‐reactive epitopes and were differentially responsive to galectin‐1 exposure. Indeed, Th1 cells were more susceptible to galectin‐1‐induced death than Th2 cells. Th2 protection from apoptosis was correlated with expression of anti‐apoptotic galectin‐3. Further, galectin‐1 promoted TCR‐induced type 2 cytokine production by Th2 cells. Differentiated Th2 cells constitutively expressed high levels of galectin‐1 and can be induced to produce even higher levels of galectin‐1 with restimulation, whereas comparable levels of galectin‐1 in Th1 cells were only observed after restimulation. Co‐culturing experiments using galectin‐1−/− and galectin‐1+/+ Th1 and Th2 T cells demonstrated that Th2‐derived galectin‐1 induced Th1 apoptosis, whereas Th1‐derived galectin‐1 promoted Th2 cytokine production. These studies identify galectin‐1 as a cross‐regulatory cytokine that selectively antagonizes Th1 survival, while promoting TCR‐induced Th2 cytokine production.

Genomic Ly49A Transgenes: Basis of Variegated Ly49A Gene Expression and Identification of a Critical Regulatory Element

Several gene families are known in which member genes are expressed in variegated patterns in differentiated cell types. Mechanisms responsible for imposition of a variegated pattern of gene expression are unknown. Members of the closely linked Ly49 inhibitory receptor gene family are expressed in a variegated fashion by NK cells. Variegated expression of these genes results in subsets of NK cells that differ in specificity for MHC class I molecules. To address the mechanisms underlying variegation, a 30-kb genomic fragment containing a single Ly49 gene was used to generate a panel of murine transgenic lines. The results demonstrated that, in almost all of the lines, the isolated Ly49A gene was expressed in a variegated pattern, remarkably similar in nearly all respects to the expression pattern of the endogenous Ly49A gene. Furthermore, the developmental timing of gene expression and regulation by host MHC molecules closely mirrored that of the endogenous Ly49A gene. Therefore, Ly49 variegation does not require competition in cis between different Ly49 genes, and the sequences imposing variegation are located proximally to Ly49 genes. Efforts to define regulatory elements of the Ly49A gene led to the identification of a DNase I hypersensitive site 4.5 kb upstream of the Ly49A gene transcription initiation site, which was shown to be essential for transgene expression. Highly related sequence elements were found upstream of other Ly49 genes, suggesting that a similar regulatory element controls each Ly49 gene.

Galectin-1 Tunes TCR Binding and Signal Transduction to Regulate CD8 Burst Size

T cell burst size is regulated by the duration of TCR engagement and balanced control of Ag-induced activation, expansion, and apoptosis. We found that galectin-1-deficient CD8 T cells undergo greater cell division in response to TCR stimulation, with fewer dividing cells undergoing apoptosis. TCR-induced ERK signaling was sustained in activated galectin-1-deficient CD8 T cells and antagonized by recombinant galectin-1, indicating galectin-1 modulates TCR feed-forward/feedback loops involved in signal discrimination and procession. Furthermore, recombinant galectin-1 antagonized binding of agonist tetramers to the TCR on activated OT-1 T cells. Finally, galectin-1 produced by activated Ag-specific CD8 T cells negatively regulated burst size and TCR avidity in vivo. Therefore, galectin-1, inducibly expressed by activated CD8 T cells, functions as an autocrine negative regulator of peripheral CD8 T cell TCR binding, signal transduction, and burst size. Together with recent findings demonstrating that gal-1 promotes binding of agonist tetramers to the TCR of OT-1 thymocytes, these studies identify galectin-1 as a tuner of TCR binding, signaling, and functional fate determination that can differentially specify outcome, depending on the developmental and activation stage of the T cell.

Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

TCR engagement triggers the polarized recruitment of membrane, actin, and transducer assemblies within the T cell–APC contact that amplify and specify signaling cascades and T effector activity. We report that caveolin-1, a scaffold that regulates polarity and signaling in nonlymphoid cells, is required for optimal TCR-induced actin polymerization, synaptic membrane raft polarity, and function in CD8, but not CD4, T cells. In CD8+ T cells, caveolin-1 ablation selectively impaired TCR-induced NFAT-dependent NFATc1 and cytokine gene expression, whereas caveolin-1 re-expression promoted NFATc1 gene expression. Alternatively, caveolin-1 ablation did not affect TCR-induced NF-κB–dependent Iκbα expression. Cav-1−/− mice did not efficiently promote CD8 immunity to lymphocytic choriomeningitis virus, nor did cav-1−/− OT-1+ CD8+ T cells efficiently respond to Listeria monocytogenes-OVA after transfer into wild-type hosts. Therefore, caveolin-1 is a T cell-intrinsic orchestrator of TCR-mediated membrane polarity and signal specificity selectively employed by CD8 T cells to customize TCR responsiveness.