VERMONT COBRE: PROJECT 1: INKT CELL ACTIVATION BY CD ID & ITS LIGANDS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Invariant natural killer T (iNKT) cells comprise a novel subset of T cells that has a profound effect on infectious disease, autoimmunity, tolerance induction, and tumor immunology. Their relatively high frequency in the gut, liver, and lung, together with their ability to rapidly secrete large amounts of a wide variety of cytokines and to activate other leukocyte subsets suggests that they may play an important role in early events of a developing immune response. All human iNKT cells express an unusual invariant T cell receptor that recognizes glycolipids derived from bacterial cell walls. In addition to these exogenous glycolipid ligands, it has been clear for some time that iNKT cells can recognize mammalian endogenous CD1d ligands. The identity, however, of natural activating ligands in mammals has remained elusive. Preliminary data from our laboratory suggests that human iNKT cells may be able to differentiate endogenous CD1d ligands from different cells. Our hypothesis is that this specificity is mediated by the iNKT TCR Vb, since the TCRa chain is invariant. This was supported by site-directed mutagenesis studies in a single amino acid change in the CDR3b loop significantly affected TCR recognition of CD1d loaded with endogenous ligand. To directly assess the contribution of iNKT TCRs to CD1d/endogenous ligand recognition, we have been developing a recombinant soluble iNKT TCR that can be multimerized to produce a high-affinity reagent with which we can directly assess relative TCR binding affinity to CD1d and its ligands using a fluorescence based approach. This "TCR tetramer" can be used in relatively straightforward binding assays directly on CD1d-expressing cells to calculate the relative Kd of different NKT TCR:CD1d ligand combinations. It would also complement and enhance approaches proposed to define biosynthetic pathways and the heterogeneity of glycosylceramides used in the formation of natural CD1d ligands and/or their precursors. For example, one potential strategy would be to use an iNKT TCR tetramer to FACS-sort CD1d+ cells that have been rendered deficient in endogenous ligand production, either through RNAi or through chemical mutagenesis. In the last year, we successfully expressed both the TCRa and b chains from two iNKT TCRs for use in these experiments. However, we were unable to successfully refold these TCRs using standard methodology. We have, therefore, now modified our design which now employs fos/jun dimerization on the C-terminus of the TCR chains to increase the level of heterodimerization. This approach has been successfully used for years in the facilitation of MHC class II heterodimer formation. We have successfully cloned these new constructs into expression vectors, expressed the individual recombinant chains as inclusion bodies, and are awaiting our first results from protein refolds. We have also made substantial headway on our second Aim, which is to dissect the biosynthetic pathways necessary for endogenous glycolipid formation. Since it is well known that mouse endogenous ligands are dependent on glucosylceramide synthase, we have been developing a targeting strategy in this species as a positive control. We have been able to successfully use an RNAi strategy to target this enzyme (the first in the GSL biosynthesis series). Furthermore, we have interesting preliminary data to suggest that, in contrast to the mouse, the human endogenous CD1d ligand may not be a glycosphingolipid. The third area of interest is our observation that there are dramatic strain differences in the iNKT cell number and function. These strain-dependent differences are correlated with significant physiological effects such as shared susceptibility to aGalCer-induced airway hyperreactivity (a cardinal feature of asthma), aGalCer-induced liver injury, and aGalCer-induced pregnancy loss. Given the ability of NKT cells to dramatically alter and shape developing immune responses, we hypothesize that variability in NKT cell number and function could be a major determinant in strain-dependent variability in resistance to certain pathogens. These observations are now in press. We have extended this work by identifying the 129/SvImJ strain as one of a few strains with severely impaired responses to aGalCer, which should prove valuable in identifying the determinants underlying these strain-dependent differences. We are now completing a thorough characterization of this strain in anticipation of its use in a congenic mapping strategy to identify loci involved in strain-dependent NKT cell function variability. Publications 2007: Rymarchyk, S.L., H. Lowenstein, J. Mayette, S.R. Foster, D. Damby, I.W. Howe, I. Aktan, R.E. Meyer, M.E. Poynter, and J.E. Boyson. Widespread natural variation in NKT cell number and function. Immunology (in press). J.E. Boyson, I. Aktan, D. Barkhuff, and A. Chant. NKT cells at the maternal-fetal interface. (submitted). Olson Jr., C.M., Bates, T.C., Radolf, J.D., Huber, S.A., Boyson, J.E., and J. Anguita. Local regulation of interferon gamma by invariant natural killer T cells modulates acute lyme carditis. (submitted). Mentoring Summaries: Elizabeth Bonney I currently meet periodically with all trainees, and discuss science as well as help with reports, presentations, and publications. I moreover participate in general career advising. Jon Boyson, a COBRE Junior Faculty is a co-investigator on my grant, "Placental immunity to LCMV" HD047224. We are also collaborating on production of Tetramers to detect NKT cells and LCMV-specific CD8 T cells. In addition, I am a consulting collaborator on his recent application: CD1D-Restricted T cells and Pregnancy Loss R01AI067897 My goal this year is to help Jon receive R21 level funding for his COBRE project, and then late help with preparation of the related R01 Sally Huber During the last year, I met with Jon twice to discuss a potential collaboration to determine cross-talk between gamma-delta T cells and Natural Killer T cells, both of which recognize CD1d, a non-classical major histocompatibility complex class I like molecule. Both NKT and gamma-delta T cells are innate effectors and modulate developing adaptive immunity in virus infection. The question to be addressed is whether they synergize or antagonize each other. I also provided help on how to write his first Progress Report on his NIH RO1 grant. Jon is currently in his second year of his RO1 and is on track to be phased out of the COBRE this year. Presumably, I would then be assigned a new mentee.