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Immunohistochemical studies have shown that a unique immunoreactive molecule is present near the apical region of human biliary epithelial (BE) cells in patients with primary biliary cirrhosis (PBC). This can be visualized by confocal microscopy in PBC livers using a number of unique monoclonal antibodies to the E2 component of pyruvate dehydrogenase complex (PDC-E2), the autoantigen most commonly recognized by antimitochondrial antibodies (AMA). One such antibody, the murine mAb C355.1 was used to identify peptide mimotopes of PDC-E2 by screening a random dodecapeptide phage library ON 159.2 to identify the possible biochemical nature of this apical staining molecule. Out of 36 independent clones, 29 showed a common sequence and seven other sequences were singly represented. Three common amino acid motifs (SYP, TYVS and VRH) were found among these eight sequences. Similar to C355.1, the human combinatorial antibodies derived from a patient with PBC, SP1 and SP4, recognize the inner lipoyl domain of PDC-E2. However, when these antibodies are used to stain PBC BE cells, SP4 stains the apical region of PBC BE cells with high intensity whereas SP1 produces only cytoplasmic staining. Competitive inhibition of immunohistochemical staining using PDC-E2 specific human combinatorial antibodies SP1 and SP4 was performed using five of the above dodecapeptides. Interestingly, the peptides selected with C355.1 differentially inhibited the binding of SP1 and SP4 to PBC BE cells. Finally, rabbit sera raised against one such peptide (WMSYPDRTLRTS) stained BE cells from patients with PBC with a higher intensity than controls. Comparable data was obtained with immunoelectronmicroscopy. These data suggest that a molecular mimic of PDC-E2 is present at the external aspect of PBC BE cells.

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

Oligomeric N-methyl D-aspartate receptor (NMDAR) in brain is a ligand-gated ion channel that becomes selectively permeable to ions upon binding to ligands. For NMDAR channel, the binding of glutamate and glycine results in opening of the calcium permeable channel. Because the calcium influx mediated by NMDAR is important for synaptic plasticity and excitotoxicity, the function of NMDA receptors has been implicated in both health and disease. Native NMDA receptors are thought to be heteromeric pentamers with a central ion conduction pathway. There are five genes (NR1, 2A, 2B, 2C, and 2D) encoding various subunits that have been cloned, and NR1 is thought to be the essential subunit since it forms a functional channel by itself. To study NMDAR structure and function, we have searched for peptide modulators of NR1 using random peptide bacteriophage libraries. The peptides were identified based on their specific association with a purified receptor fusion protein that contains the putative ligand binding domain. We report the identification of one group of cyclic peptides (Mag-1) with a consensus sequence of CDGLRHMWFC. Using biochemical binding analysis and patch clamp electrophysiological recording, we show that the synthetic Mag-1 peptides cause noncompetitive inhibition of the receptor channel activity.

In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.

The lineage of NK cells and their relationship to T lymphocytes have been controversial issues. Since rearrangement of the T cell antigen receptor beta chain genes occurs early in the ontogeny and differentiation of all T cells, this can be used as an unequivocal marker to discriminate T from non-T lymphocytes. Recent studies (16-18) examining T cell antigen receptor gene rearrangement and expression in certain IL-2-dependent NK cell lines and leukemias have revealed that some lines rearrange C beta genes, whereas others do not. However, it is important to establish whether these cell lines are representative of the major population of NK cells freshly derived from the host. Herein, we have purified granulocytes, CD16+ NK cells and T lymphocytes from human peripheral blood, prepared genomic DNA from each cell type, and then examined the organization of their T cell antigen receptor genes by restriction enzyme analysis using a C beta cDNA as probe. The C beta genes were in germline configuration in NK cells and granulocytes. In contrast, peripheral blood T lymphocytes showed rearrangement of the C beta gene. These data support the hypothesis that the majority of human peripheral blood NK cells are fundamentally distinct from T lymphocytes in lineage and nonself recognition.

Cytotoxicity not restricted by the major histocompatibility complex (MHC) is mediated by two distinct types of lymphocyte: natural killer (NK) cells and non-MHC-restricted cytotoxic T lymphocytes (CTL). These two types of cytotoxic lymphocytes can be distinguished by antigenic phenotype, function, and molecular genetic studies. In human peripheral blood, NK cells are identified by expression of the Leu-19 and/or CD16 cell surface antigens, and lack of CD3/T cell antigen receptor (Ti) complex expression (i.e., CD3-,Leu-19+). Peripheral blood non-MHC-restricted CTL express both CD3 and Leu-19 (i.e., CD3+, Leu-19+, referred to as Leu-19+ T cells). Both Leu-19+ T cells and NK cells lyse "NK-sensitive" hematopoietic tumor cell targets, such as K562, without deliberate immunization of the host. However, most "NK activity" in peripheral blood is mediated by NK cells, because they are usually more abundant and more efficient cytotoxic effectors than Leu-19+ T cells. The cytolytic activity of both NK cells and Leu-19+ T cells against hematopoietic targets was enhanced by recombinant interleukin 2 (rIL 2). NK cells, but not peripheral blood Leu-19+ T cells, were also capable of lysing solid tumor cell targets after short-term culture in rIL 2. Southern blot analysis of NK cells revealed that both the T cell antigen receptor beta-chain genes and the T cell-associated gamma genes were not rearranged, but were in germ-line configuration. These findings indicate that NK cells are distinct in lineage from T lymphocytes and do not use the T cell antigen receptor genes for target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study we have identified and characterized three subpopulations of peripheral blood NK cells based on the surface expression of CD56 and CD16. We have designated these subsets CD16neg, CD16dim, and CD16bright according to the relative surface density of CD16. The CD16bright subset comprised about 10% to 15% of PBL, whereas the CD16dim and CD16neg subsets comprise less than 1% of the total lymphocytes. A detailed characterization of these subsets revealed both similarities and differences. The three subsets shared a great deal of phenotypic similarity, expressing CD2, CD7, CD11b, CD38, CD45R, CD18, and the p75 IL-2R on the majority of the cells in each subset. There were, however, several prominent phenotypic differences, particularly in the expression of CD57, CD11c, CD44, CD25, Leu-8, L263, and L265. The CD16neg cells were morphologically large agranular lymphocytes and demonstrated low levels of non-MHC restricted cytolysis of NK-sensitive tumor lines. The CD16dim and CD16bright subsets were large granular lymphocytes and revealed potent cytotoxicity against NK-sensitive targets. All subsets demonstrated IL-2-dependent activation and proliferation; however, the CD16dim and CD16neg subsets were preferentially responsive to very low concentrations of rIL-2. Although rIL-4 effectively inhibited the IL-2-induced cytolytic activation of all three NK cell subsets, only the CD16bright cells showed rIL-4 inhibition of IL-2 dependent proliferation. Cytokine transcription was also differentially regulated in the NK cell subsets after rIL-2 activation. Although TNF-alpha was equally transcribed in each subsets, IFN-gamma and serine protease-HF were preferentially transcribed in the CD16bright NK cells. Based on these results, we propose that these NK cell subsets represent portions of the NK cell differentiation pathway present in the peripheral blood.

The locus of the delta chain of the human T-cell receptor has been isolated and examined. Three D (diversity) regions and two J (joining) regions are present on the 5' side of the C (constant) region. The closest V (variable) region to the constant region is V delta 2, which in the germ line is found on the 3' side of the constant region in an inverted direction. The genomic structure of the human locus closely parallels its mouse counterpart. Several cDNA sequences and a series of rearranged genomic sequences are compared which demonstrate an enormous potential diversity in the junctional region, between the variable region and the joining region. We find the predominant utilization of the PEER variable region in thymic polyclonal gamma delta cell lines and in some peripheral blood gamma delta cell lines. Thus, the delta chain may have relatively limited variable-region diversity but a large junctional-region diversity. The implications of this observation are discussed.

CD16 is a low-affinity immunoglobulin G (IgG) Fc receptor that is expressed on natural killer (NK) cells, granulocytes, activated macrophages, and some T lymphocytes. Two similar genes, CD16-I and CD16-II, encode membrane glycoproteins that are anchored by phosphatidylinositol (PI)-glycan and transmembrane polypeptides, respectively. The primary structural requirements for PI-linkage were examined by constructing a series of hybrid cDNA molecules. Although both cDNA's have an identical COOH-terminal hydrophobic segment, CD16-I has Ser203 whereas CD16-II has Phe203. Conversion of Phe to Ser in CD16-II permits expression of a PI-glycan-anchored glycoprotein, whereas conversion of Ser to Phe in CD16-I prevents PI-glycan linkage.

The gamma-TCR is encoded by genes composed of V, J, and C elements that demonstrate a limited potential for recombinational diversity. These genes are rearranged, transcribed, and translated into proteins early during thymic ontogeny. Lymphocytes express gamma-TCR proteins on the plasma membrane only in association with the CD3 complex. gamma-TCR glycoproteins usually associate with another non-gamma glycoprotein, designated delta-TCR, to form a heterodimer receptor. Both non-disulfide-bonded and disulfide-bonded gamma/delta-TCR heterodimers have been identified on the plasma membrane of human T lymphocytes. On certain gamma-TCR-bearing T cell lines, a delta-TCR protein cannot be visualized by autoradiography. It is possible that delta-TCR proteins are associated with gamma-TCR glycoproteins on these cell lines but are not efficiently radiolabeled. Alternatively, it has been suggested that homodimers of gamma-TCR proteins can assemble with CD3 and be expressed on the plasma membrane of these cells. In adult lymphoid tissues, the majority of T lymphocytes expresses a CD3, alpha/beta antigen receptor, whereas only a minor subset (less than 5% of peripheral blood lymphocytes, lymph node, spleen, and thymocytes) express a CD3, gamma/delta antigen receptor. IL-2-dependent cell lines of both murine and human CD3, gamma/delta T cells have been established. Most CD3, gamma/delta T cell lines mediate cytotoxicity against a broad spectrum of tumor-cell targets, although the functional significance of this observation remains unclear. Cytotoxicity is apparently not restricted by or directed against MHC antigens. Antibodies against CD3 or gamma-TCR can induce proliferation and IL-2 secretion and can either augment or inhibit cytotoxicity, demonstrating that the gamma/delta-TCR is a functional receptor. The ligand recognized by this receptor has not been identified. The physiological role of T lymphocytes expressing gamma/delta-TCR, the molecular and structural properties of delta-TCR, and the relationship between CD3, alpha/beta T lymphocytes and CD3, gamma/delta T lymphocytes are the major unresolved questions that will be the primary focus of further experimentation.