Honoré Mazarguil

T Cells Infiltrate the Brain in Murine and Human Transmissible Spongiform Encephalopathies

ABSTRACT CD4 and CD8 T lymphocytes infiltrate the parenchyma of mouse brains several weeks after intracerebral, intraperitoneal, or oral inoculation with the Chandler strain of mouse scrapie, a pattern not seen with inoculation of prion protein knockout (PrP−/−) mice. Associated with this cellular infiltration are expression of MHC class I and II molecules and elevation in levels of the T-cell chemokines, especially macrophage inflammatory protein 1β, IFN-γ-inducible protein 10, and RANTES. T cells were also found in the central nervous system (CNS) in five of six patients with Creutzfeldt-Jakob disease. T cells harvested from brains and spleens of scrapie-infected mice were analyzed using a newly identified mouse PrP (mPrP) peptide bearing the canonical binding motifs to major histocompatibility complex (MHC) class I H-2b or H-2d molecules, appropriate MHC class I tetramers made to include these peptides, and CD4 and CD8 T cells stimulated with 15-mer overlapping peptides covering the whole mPrP. Minimal to modest Kb tetramer binding of mPrP amino acids (aa) 2 to 9, aa 152 to 160, and aa 232 to 241 was observed, but such tetramer-binding lymphocytes as well as CD4 and CD8 lymphocytes incubated with the full repertoire of mPrP peptides failed to synthesize intracellular gamma interferon (IFN-γ) or tumor necrosis factor alpha (TNF-α) cytokines and were unable to lyse PrP−/− embryo fibroblasts or macrophages coated with 51Cr-labeled mPrP peptide. These results suggest that the expression of PrPsc in the CNS is associated with release of chemokines and, as shown previously, cytokines that attract and retain PrP-activated T cells and, quite likely, bystander activated T cells that have migrated from the periphery into the CNS. However, these CD4 and CD8 T cells are defective in such an effector function(s) as IFN-γ and TNF-α expression or release or lytic activity.

Identification of neuropeptide FF-related peptides in rodent spinal cord

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

Direct identification of a peptide binding region in the ORL1 receptor by photo-affinity labelling with [Bpa10, Tyr14]nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe10,Tyr14]nociceptin ([Bpa10,Tyr14]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa10,Tyr14]noc binds the receptor with high affinity (K i ∼0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC50 ∼0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa10,Tyr14]noc results in the irreversible labeling of a glycoprotein of ∼65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (∼6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of ∼3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu295 → Asp mutant receptor also yields a single labeled fragment of ∼6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296–302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa10]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile300 within the experimentally identified photoreactive region.

Genetically Encoded and Post-translationally Modified Forms of a Major Histocompatibility Complex Class I-restricted Antigen Bearing a Glycosylation Motif Are Independently Processed and Co-presented to Cytotoxic T Lymphocytes

The mechanisms by which antigenic peptides bearing a glycosylation site may be processed from viral glycoproteins, post-translationally modified, and presented by major histocompatibility complex class I molecules remain poorly understood. With the aim of exploring these processes, we have dissected the structural and functional properties of the MHC-restricted peptide GP92–101 (CSANNSHHYI) generated from the lymphocytic choriomeningitis virus (LCMV) GP1 glycoprotein. LCMV GP92–101 bears a glycosylation motif -NXS- that is naturallyN-glycosylated in the mature viral glycoprotein, displays high affinity for H-2Db molecules, and elicits a CD8+ cytotoxic T lymphocyte response. By analyzing the functional properties of natural and synthetic peptides and by identifying the viral sequence(s) from the pool of naturally occurring peptides, we demonstrated that multiple forms of LCMV GP92–101 were generated from the viral glycoprotein and co-presented at the surface of LCMV-infected cells. They corresponded to non-glycosylated and post-translationally modified sequences (conversion of Asn-95 to Asp or alteration of Cys-92). The glycosylated form, despite its potential immunogenicity, was not detected. These data illustrate that distinct, non-mutually exclusive antigen presentation pathways may occur simultaneously within a cell to generate structurally and functionally different peptides from a single genetically encoded sequence, thus contributing to increasing the diversity of the T cell repertoire.