A functional comparison of the domestic cat bitter receptors Tas2r38 and Tas2r43 with their human orthologs.

BACKGROUND: Domestic cats (felis catus) have a reputation for being rather unpredictable in their dietary choices. While their appetite for protein or savory flavors is consistent with their nutritional needs, their preference among protein-sufficient dietary options may relate to differences in the response to other flavor characteristics. Studies of domestic cat taste perception are limited, in part, due to the lack of receptor sequence information. Several studies have described the phylogenetic relationship of specific cat taste receptor sequences as compared with other carnivores. For example, domestic cats are obligate carnivores and their receptor Tas1r2, associated with the human perception of sweet, is present only as a pseudogene. Similarly, the cat perception of bitter may differ from that of other mammals due to variations in their repertoire of bitter receptor (Tas2r) genes. This report includes the first functional characterization of domestic cat taste receptors. RESULTS: We functionally expressed two uncharacterized domestic sequences Tas2r38 and Tas2r43 and deorphanized the receptors using a cellular functional assay. Statistical significance was determined using an unpaired, two-tailed t-test. The cat sequence for Tas2r38 contains 3 major amino acid residues known to confer the taster phenotype (PAI), which is associated with sensitivity to the bitter compounds PROP and PTC. However, in contrast to human TAS2R38, cat Tas2r38 is activated by PTC but not by PROP. Furthermore, like its human counterpart, cat Tas2r43 is activated by aloin and denatonium, but differs from the human TAS2R43 by insensitivity to saccharin. The responses of both cat receptors to the bitter ligands were concentration-dependent and were inhibited by the human bitter blocker probenecid. CONCLUSIONS: These data demonstrate that the response profiles of the cat bitter receptors Tas2r38 and Tas2r43 are distinct from those of their orthologous human receptors. Results with cat Tas2r38 also demonstrate that additional residues beyond those classically associated with PROP sensitivity in humans influence the sensitivity to PROP and PTC. Functional studies of the human bitter receptor family are being applied to the development of food and medicinal products with more appealing flavor profiles. Our work lays the foundation for similar work applied to felines.

CD8α(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites.

CD8α(+) dendritic cells (DCs) are important in vivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, secretion of interleukin-12 (IL-12) by CD8α(+) DCs suggests a role for these cells in response to Toxoplasma gondii antigens, although it remains unclear whether these cells are required for protection against T. gondii infection. Toward this goal, we examined T. gondii infection of Batf3(-/-) mice, which selectively lack only lymphoid-resident CD8α(+) DCs and related peripheral CD103(+) DCs. Batf3(-/-) mice were extremely susceptible to T. gondii infection, with decreased production of IL-12 and interferon-γ. IL-12 administration restored resistance in Batf3(-/-) mice, and mice in which IL-12 production was ablated only from CD8α(+) DCs failed to control infection. These results reveal that the function of CD8α(+) DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T. gondii infection.

Targeting of B and T lymphocyte associated (BTLA) prevents graft-versus-host disease without global immunosuppression.

Graft-versus-host disease (GVHD) causes significant morbidity and mortality in allogeneic hematopoietic stem cell transplantation (aHSCT), preventing its broader application to non-life-threatening diseases. We show that a single administration of a nondepleting monoclonal antibody specific for the coinhibitory immunoglobulin receptor, B and T lymphocyte associated (BTLA), permanently prevented GVHD when administered at the time of aHSCT. Once GVHD was established, anti-BTLA treatment was unable to reverse disease, suggesting that its mechanism occurs early after aHSCT. Anti-BTLA treatment prevented GVHD independently of its ligand, the costimulatory tumor necrosis factor receptor herpesvirus entry mediator (HVEM), and required BTLA expression by donor-derived T cells. Furthermore, anti-BTLA treatment led to the relative inhibition of CD4(+) forkhead box P3(-) (Foxp3(-)) effector T cell (T eff cell) expansion compared with precommitted naturally occurring donor-derived CD4(+) Foxp3(+) regulatory T cell (T reg cell) and allowed for graft-versus-tumor (GVT) effects as well as robust responses to pathogens. These results suggest that BTLA agonism rebalances T cell expansion in lymphopenic hosts after aHSCT, thereby preventing GVHD without global immunosuppression. Thus, targeting BTLA with a monoclonal antibody at the initiation of aHSCT therapy might reduce limitations imposed by histocompatibility and allow broader application to treatment of non-life-threatening diseases.

IL-15 regulates both quantitative and qualitative features of the memory CD8 T cell pool.

Memory T cells are critical for immunity to various intracellular pathogens. Recent studies have indicated that CD8 secondary memory cells, induced by prime-boost approaches, show enhanced protective function compared with primary memory cells and exhibit phenotypic and functional characteristics that distinguish them from primary memory cells. However, little is known about the cytokine requirements for generation and maintenance of boosted memory CD8 T cells. We studied the role of IL-15 in determining the size and composition of the secondary (2 degrees) memory CD8 T cell pool induced by Listeria monocytogenes infection in mice. Following boosting, IL-15-deficient animals failed to generate a subset of CD8 effector memory cells, including a population of IL-7Ralpha(low) cells, which were prominent among secondary memory cells in normal mice. IL-15 deficiency also resulted in changes within the IL-7Ralpha(high)CD62L(low) subset of 2 degrees memory CD8 T cells, which expressed high levels of CD27 but minimal granzyme B. In addition to these qualitative changes, IL-15 deficiency resulted in reduced cell cycle and impaired Bcl-2 expression by 2 degrees memory CD8 T cells, suggesting a role for IL-15 in supporting both basal proliferation and survival of the pool. Analogous qualitative differences in memory CD8 T cell populations were observed following a primary response to Sendai virus in IL-15(-/-) animals. Collectively, these findings demonstrate that IL-15 plays an important role in dictating the composition rather than simply the maintenance of the CD8 memory pool.

Transcription factor Mef2c is required for B cell proliferation and survival after antigen receptor stimulation.

Calcineurin is required for B cell receptor (BCR)-induced proliferation of mature B cells. Paradoxically, loss of NFAT transcription factors, themselves calcineurin targets, induces hyperactivity, which suggests that calcineurin targets other than NFAT are required for BCR-induced proliferation. Here we demonstrate a function for the calcineurin-regulated transcription factor Mef2c in B cells. BCR-induced calcium mobilization was intact after Mef2c deletion, but loss of Mef2c caused defects in B cell proliferation and survival after BCR stimulation in vitro and lower T cell-dependent antibody responses and germinal center formation in vivo. Mef2c activity was specific to BCR stimulation, as Toll-like receptor and CD40 signaling induced normal responses in Mef2c-deficient B cells. Mef2c-dependent targets included the genes encoding cyclin D2 and the prosurvival factor Bcl-x(L). Our results emphasize an unrecognized but critical function for Mef2c in BCR signaling.

IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells.

Naive T cells undergo slow homeostatic proliferation in response to T cell lymphopenia, which is also called lymphopenia-induced proliferation (LIP). IL-7 is critically required for this process, but previous studies suggested IL-15 was expendable for LIP of naive CD8 T cells. In contrast, we show that IL-15 is important for sustained CD8 T cell proliferation and accumulation in a lymphopenic setting, as revealed by truncated LIP in IL-15(-/-) hosts. At the same time, we find that IL-12 enhances LIP by acting directly on the CD8 T cells and independently of IL-15, suggesting distinct pathways by which cytokines can regulate homeostatic proliferation. Interestingly, the memory-phenotype CD8 T cell generated by LIP in IL-15(-/-) hosts are phenotypically distinct from the rare endogenous memory-phenotype cells found in IL-15(-/-) animals, suggesting these cells are generated by different means. These findings demonstrate that cytokine requirements for LIP change during the process itself, illustrating the need to identify factors that regulate successive stages of lymphopenia-driven proliferation.

Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18.

Production of IFN-gamma by CD4 T cells is generally thought to be mediated by TCR triggering, however, Ag-nonspecific activation of effector CD8 T cells has been reported in infection models. In this study, we demonstrate that Ag-experienced CD4 T cells in the spleen of Salmonella-infected mice acquire the capacity to rapidly secrete IFN-gamma in response to stimulation with bacterial lysate or LPS. This innate responsiveness of T cells was transient and most apparent during, and immediately following, active Salmonella infection. Furthermore, innate T cell production of IFN-gamma in response to bacterial lysate or LPS was Ag independent and could be induced in Listeria-infected mice and in the absence of MHC class II expression. IL-18 was required for maximal innate responsiveness of CD4 T cells in Salmonella-infected mice and for optimal bacterial clearance in vivo. These data demonstrate that CD4 T cells acquire the capacity to respond to innate stimuli during active bacterial infection, a process that may contribute significantly to amplifying effector responses in vivo.

The sialyltransferase ST3Gal-I is not required for regulation of CD8-class I MHC binding during T cell development.

The CD8 coreceptor plays a crucial role in thymocyte and T cell sensitivity by binding to class I MHC and recruiting downstream signaling molecules to the TCR. Previous studies reported considerable changes in TCR-independent CD8/class I MHC binding (i.e., CD8 noncognate interactions) during T cell development, changes that correlated with altered glycosylation of surface molecules. In particular, expression of the sialyltransferase ST3Gal-I has been proposed as a critical factor regulating the attenuation of CD8 avidity during the double-positive to CD8 single-positive progression. This hypothesis is strengthened by the fact that ST3Gal-I(-/-) animals show a profound disregulation of CD8 T cell homeostasis. In contrast to this model, however, we report in this study that ST3Gal-I deficiency had no detectable impact on CD8 noncognate binding to multimeric peptide/MHC class I ligands at any stage of thymocyte development. We also found that the susceptibility to CD8-induced cell death is not markedly influenced by ST3Gal-I deficiency. Thus, the profound effects of ST3Gal-I on CD8 T cell survival evidently do not involve a role for this enzyme in controlling CD8-class I binding.

The timing of TCR alpha expression critically influences T cell development and selection.

Sequential rearrangement of the T cell receptor for antigen (TCR) beta and alpha chains is a hallmark of thymocyte development. This temporal control is lost in TCR transgenics because the alpha chain is expressed prematurely at the CD4- CD8- double negative (DN) stage. To test the importance of this, we expressed the HY alpha chain at the physiological CD4+ CD8+ double positive (DP) stage. The reduced DP and increased DN cellularity typically seen in TCR transgenics was not observed when the alpha chain was expressed at the appropriate stage. Surprisingly, antigen-driven selection events were also altered. In male mice, thymocyte deletion now occurred at the single positive or medullary stage. In addition, no expansion of CD8 alpha alpha intestinal intraepithelial lymphocytes (IELs) was observed, despite the fact that HY transgenics have been used to model IEL development. Collectively, these data establish the importance of proper timing of TCR expression in thymic development and selection and emphasize the need to use models that most accurately reflect the physiologic process.

Cutting edge: transpresentation of IL-15 by bone marrow-derived cells necessitates expression of IL-15 and IL-15R alpha by the same cells.

IL-15 is critical for generation of multiple lymphoid subsets. Recent data have demonstrated a unique aspect of responses to IL-15, in that cells bearing the IL-15Ralpha chain can bind soluble IL-15 and "transpresent" the cytokine to other cells, allowing the latter to respond to IL-15. However, it is unclear whether IL-15 is normally secreted and then becomes bound to surface IL-15Ralpha on bystander cells, or whether transpresentation is mediated by the same cells which synthesize IL-15. Using mixed bone marrow chimeric mice, we present evidence for the latter model, showing that development of NK cells and memory phenotype CD8 T cells necessitates that both IL-15 and IL-15Ralpha be expressed by the same population of cells. These data argue that soluble forms of IL-15 are irrelevant for physiological responses to this cytokine, and the implications of this finding are discussed.