The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme factions obtained from light-treated and dark-grown wid type, albino-i, alZbi-2, albino-3, and white collar-I strains were mixed in various combinations, and the activity for conversion of 11-'4Clisopen-tenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo lght treatment increases the enzyme actvity in the particulate fraction; (d) this light effect occurs in wild type, albno-I, and albin-3 straiw and (e) enzyme activity is present in the particulate faction obtained from the white collar-i mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity,

Identification of the targets of autoreactive T cells is important for understanding the pathogenesis of many autoimmune diseases. In multiple sclerosis, myelin proteins are thought to be the targets of autoreactive T-cell responses. To date only major histocompatibility complex class 11-restricted CD4+ T-cell responses to myelin proteins have been investigated. In the present study, the ability of self peptides derived from human myelin proteins to induce autoreactive CD8+ T-cell responses has been assessed. Peptide sequences from human myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin oligodendrocyte glycoprotein have been identified that bind to and form stable complexes with HLA-A2. MBP 110-118, PLP 80-88, MAG 287-295, MAG 509-517, and MAG 556-564 were all able to induce peptide-specific HLA-A2-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro in HLA-A2+ individuals. CTLs specific for MBP 110-118 and MAG 556-564 could recognize endogenously processed antigens presented by HLA-A2. CTL clones reactive to MBP 110-118 and MAG 556-564 produced tumor necrosis factor a and a subset ofthese clones also produced interferon y. These results demonstrate that (i) self peptides derived from human myelin proteins can induce autoreactive CD8+ CTLs and (ii) these CD8+ T cells produce cytokines thought to be important in mediating demyelinating disease. These studies provide an experimental approach for the assessment of CD8+ T-cell responses in such autoimmune diseases.sively by MHC class I molecules (17, 18), and adult oligodendrocytes constitutively express MHC class I molecules but do not express class II MHC molecules, even when stimulated by interferon y (IFN-y) (19). Thus, if adult oligodendrocytes are targets for T-cell reactivity, they will most likely be recognized by MHC class I-restricted CD8+ T cells but not by class II-restricted CD41 T cells. (iii) In an animal model of demyelinating disease, experimental allergic encephalomyelitis (EAE), CD8+ T cells have been shown to have an immunoregulatory effect on the course of the disease (20, 21). Despite these implications that autoreactive CD8+ T-cell responses may play an important role in MS, a successful experimental approach to this question has not yet been reported.To investigate the possible role(s) of CD8+ T-cell responses to myelin proteins in the pathogenesis of demyelinating disorders, we have taken advantage of recent advances in the ability to predict peptide epitopes presented by the HLA class I molecule, . A computer-based algorithm has been devised that predicts the stability of HLA-A2-peptide complexes by quantitating positive and negative effects on binding by each amino acid within a nonamer peptide (22). This algorithm has been used to predict HLA-A2 binding nonamer peptides from the myelin proteins MBP, PLP, MAG, and MOG. In this report we have identified HLA-A2-restricted epitopes derived from these proteins that are capable of inducing CD8+ cytotoxic T-lymphocyte (CTL)...

Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-α/βs make multiple contacts with the α1 and α2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the α1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2–specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2–specific TCRs, there is a location on the central portion of the α1 helix that provides interactions crucial to their function with the MHC molecule.

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Most peptides that bind to a particular MHC class I molecule share amino acid residues that are thought to physically "anchor" the peptide to polymorphic pockets within the class I binding site. Sequence analysis of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. In vitro assembly assays employing synthetic peptides and recombinant HLA-B44 produced by Escherichia coli revealed that an acidic amino acid at P2 was necessary for promoting stable peptide binding to HLA-B44. Surprisingly, although Tyr was almost exclusively found at P9 of the endogenous peptide sequences, a wide variety of amino acid residues such as Leu, Ala, Arg, Lys, His, and Phe could be tolerated at this position. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. Finally, molecular models were created to investigate the possible ways in which the anchor residues might function to stabilize the binding of peptides to HLA-B44, and these models indicate that the acidic residue at P2 most likely interacts primarily with Lys 45 of the HLA-B44 heavy chain and makes additional contacts with Ser 67 and Tyr 9.

TGF-␤ isoforms are key modulators of a broad range of biological pathways and increasingly are exploited as therapeutic targets. Here, we describe the crystal structures of a pan-TGF-␤ neutralizing antibody, GC-1008, alone and in complex with TGF-␤3. The antibody is currently in clinical evaluation for idiopathic pulmonary fibrosis, melanoma, and renal cell cancer. GC-1008 recognizes an asymmetric binding interface across the TGF-␤ homodimer with high affinity. Whereas both cognate receptors, TGF-␤-receptor types I and II, are required to recognize all 3 TGF-␤ isoforms, GC-1008 has been engineered to bind with high affinity to TGF-␤1, 2, and 3 via a single interaction surface. Comparison with existing structures and models of TGF-␤ interaction with its receptors suggests that the antibody binds to a similar epitope to the 2 receptors together and is therefore a structurally different but functionally identical mimic of the binding mode of both receptors.cancer ͉ fibrotic diseases ͉ TGF-␤/antibody complex ͉ TGF-␤ signaling ͉ X-ray structure T he transforming growth factor ␤ (TGF-␤) superfamily of cytokines comprises Ͼ30 structurally related proteins that are involved in the regulation of a wide variety of biological processes such as cell proliferation, differentiation, and expression of extracellular matrix proteins (1, 2). Members of this family are Ϸ25-kDa homodimeric molecules with a similar structural framework in which the 2 monomers are covalently linked via a disulfide bridge (3-8). Three different isoforms of TGF-␤ are known in mammals that share a sequence identity of 70-82% (2). All 3 isoforms of TGF-␤ are expressed as latent or inactive propeptides that can be activated by a diverse number of physiologically and pathophysiologically associated mechanisms such as thrombospondin-1, integrin ␣v␤6, reactive oxygen species, and low pH (9, 10). Expression of TGF-␤ isoforms and the activation of latent TGF-␤ to mature active protein are tightly regulated in normal physiology, and a dysregulation of this process has been described in many pathological conditions leading to an enhanced activity of TGF-␤ in diseases such as fibrotic disease and some malignancies. Gene-deletion studies in vivo indicate that the 3 mammalian isoforms of TGF-␤ have nonoverlapping activities essential for vascular development and the regulation of immune cell function (11-13). However, in vitro, the biological activities of the 3 isoforms of TGF-␤ are almost identical. TGF-␤1 and TGF-␤3 trigger the cellular signaling cascade upon binding to the extracellular domains of 2 transmembrane receptors, known as TGF-␤ receptor types I and II, forming a ternary complex. This complex assembly occurs first through high affinity binding of the cytokines to their TGF-␤ receptor type II, followed by the recruitment of the TGF-␤ receptor type I (14, 15). The binding potency of TGF-␤ to its type II receptor is isoform-dependent, with the highest binding affinities for TGF-␤1 and 3 and a 10-to 20-fold-smaller binding affinity for 17). The formation ...

A motif specific to peptides that bind to the human class I major histocompatibility complex molecule HLA-A3 was identified by sequence analysis ofHPLC fractions containing endogenous peptides. Twenty-six different sequences were obtained, 19 of which were nonamers. The majority of these endogenous peptide sequences contained Leu at position (P)2, while most sequences contained Tyr or Lys at P9. In addition, Phe was shared by 16 sequences at P3. Synthetic peptides corresponding to endogenous peptide sequences were shown to bind to HLA-A3. The importance of Leu at P2 and Tyr or Lys at P9 ("anchor" residues) for peptide binding to HLA-A3 was demonstrated by the following results: (i) peptides GLFGGGGGY, GLFGGGGGK, and GLGGGGFGY, but not GLFGGGGGV, specifically bound to HLA-A3 and (iu) six nonapeptides from within the influenza A nucleoprotein, matrix, and polymerase proteins, selected for synthesis based upon their possession of P2 and P9 anchor residues, were shown to bind HLA-A3. In contrast, none of a set of eight peptides that bound to HLA-A2, or six that bound to HLA-B27, bound detectably to HLA-A3. These rmdings provide a rationale for the design and selection of peptides that can be recognized by HLA-A3-restricted T cells.

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