Biomedical subjects

J E Coligan

Publications and source records attributed to J E Coligan.

At least 109 records · Page 6Linked to original sources

PubMed1991

Selective autoregulation of endothelins in primary astrocyte cultures: endothelin receptor-mediated potentiation of endothelin-1 secretion.

Observations that primary rat astrocytes express high-affinity binding sites for endothelins and, in addition, are capable of producing not only endothelin-3 but also endothelin-1 prompted the investigation of a possible relation between endothelin peptides and receptors in these cells. Sarafotoxin S6b, an endothelin receptor agonist, was used as a tool to study endothelin receptor-mediated changes in the secretion of endothelin-1 and -3. The effects of sarafotoxin S6b and endothelin-1 in stimulating inositolphospholipid turnover as well as in inducing AP1 in primary astrocyte cultures were found to be similar. A low cross-reactivity of sarafotoxin S6b with endothelin-1 and -3 in the endothelin radioimmunoassays used here, along with a distinctly different elution position in high-performance liquid chromatography, allowed a clear discrimination between sarafotoxin and endothelins in the culture media. Stimulation of primary rat astrocytes with 10(-7) M sarafotoxin S6b for 1 hour resulted in a substantial increase in endothelin-1 immunoreactivity in the medium. This immunoreactivity reached a peak at 3 hours and showed no further increase after 8 and 24 hours. Treatment of our cultures with phorbol myristate acetate, lipopolysaccharide, tumor necrosis factor alpha, and norepinephrine for 24 hours led to only a moderate elevation of endothelin-1 immunoreactivity. Immunoreactive endothelin-3 was not affected by any of the treatments tested. Thus, our data suggest that endothelins in primary rat astrocytes are subject to selective autoregulation, as demonstrated by the potentiation of endothelin-1 secretion after activation of glial endothelin receptors.

Animals↗

PubMed1990

Endothelins, peptides with potent vasoactive properties, are produced by human macrophages.

Endothelins are peptides, originally isolated from endothelial cells, with potent vasoactive and mitogenic properties. In this study, we demonstrate that human macrophages synthesize and secrete endothelins. Cultured human macrophages were found by immunocytochemistry to stain positively for endothelin 1 and endothelin 3. Their capability to produce and release these peptides was confirmed by a combination of reverse-phase high-performance liquid chromatography and radioimmunoassays, specific for endothelin 1 and 3, respectively. Immunoreactive peptides were identified both in cellular extracts and in macrophage-conditioned medium. The secretion of endothelin 1, but not of endothelin 3, from macrophages could be stimulated 6-10-fold by lipopolysaccharide or phorbol myristate acetate (PMA). Northern blot analysis of total macrophage RNA using an endothelin 1 cDNA probe revealed induction of endothelin mRNA in PMA-treated macrophages. Furthermore, immunoreactive endothelin 1 and 3 were found in U937 cells, a human promonocytic line, and in freshly isolated human monocytes. In contrast, no immunoreactive endothelin was detected in cell extracts from human neutrophils and lymphocytes. The expression of endothelins in tissue macrophages was demonstrated in paraffin sections of human lung using immunohistochemistry. In conclusion, the finding that human macrophages produce endothelins suggests an important role for these peptides in the microenvironment of tissue macrophages. Macrophage-derived endothelins may have an essential function in blood vessel physiology, and aberrant production may contribute to vessel pathology.

Cells, Cultured↗

PubMed1990

T cell receptor delta-gene expression and diversity in the mouse spleen.

Thirteen T cell hybridomas and two cell lines expressing gamma delta TCR were generated from B10 and B6 CD3+CD4-CD8- splenic T cells. At least three different types of gamma-chains (V gamma 2-C gamma 1, C gamma 2, and C gamma 4) were shown to be expressed in these cells. Analysis of V delta gene segment usage in these hybridomas was performed by Northern blot hybridizations using probes specific for the V delta 1 through V delta 6 gene families and electrophoretic analysis of polymerase chain reaction amplified DNA obtained using V region-specific oligonucleotide primers for the V delta gene families as well as for V alpha 10, V alpha 4, and V alpha 11. These analyses revealed that V delta 5 gene segments are used by a high percentage of splenic gamma delta CD4-CD8- T cells (53% of the cells in our panel). Other V delta segments expressed by our panel of cells were V delta 2, V delta 4, V delta 6, and V alpha 10, indicating that the V delta repertoire expressed in the spleen is similar, but possibly not identical to the adult thymus repertoire. Sequence analysis of the V-D-J joinings of the delta-chain messages revealed substantial diversity, indicating that the delta-chain repertoire expressed in peripheral lymphoid organs uses a significant portion of the potential diversity predicted for these chains. These results demonstrate that, in spite of the low numbers of gamma delta T cells in peripheral lymphoid organs, the diversity of their TCR is extensive enough to play an important role in the immune response.

Amino Acid Sequence↗

PubMed1990

Physical characterization of histidine-rich protein from Plasmodium lophurae.

The size and shape of Plasmodium lophurae histidine-rich protein have been determined by analytical centrifugation and electron microscopy. From the partial specific volume of 0.72 cc/g, the molecular weight was determined to be 43,000. The sedimentation velocity studies indicated a coefficient of 1.32 S in 0.9 M acetic acid (pH 3.5), monodispersity and significant asymmetry. Darkfield electron microscopy revealed the major species to be compact oblate spheroids 12 nm in width and extended filamentous particles of average length 35 nm by 1.5 nm. Analysis of the sequence of the protein by the method of Garnier et al. (J. Mol. Biol. (1978) 120, 97-120) predicted that 82% of its residues would be found in three long alpha-helices. The protein's CD spectrum has a strong resemblance to that of poly(L-histidine) at pH 4-5, where the homopolymer is thought to be in a right-handed alpha-helical form. A single helix containing 300 residues would be 45 nm long, the largest length found by electron microscopy. From the electron-microscopic data, sedimentation coefficients of 1.6 and 1.95 S, respectively, were calculated for flexible-coil and extended-rod models, in closer agreement with the measured value of 1.3 S than the value calculated for a spherical model. Thus, the major species in acetic acid is probably an incompletely extended rod which, as the pH is increased to neutrality, condenses to form spherical molecular aggregates seen in the malaria parasite.

Animals↗

PubMed1990

Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor.

The epidermis of clinically normal-appearing human skin harbors a phenotypically heterogeneous population of T lymphocytes (TCs), the majority of which are CD2+/CD3+/CD5+ "memory" cells, but in an unactivated state, and express the TCR-alpha/beta. In contrast to murine skin, only a very minor subpopulation of CD3+ cells in the human epidermis bears the TCR-gamma/delta. Epidermal TCs primarily are distributed along the rete ridges in the basal keratinocyte layer and are often in close apposition to Langerhans cells (LCs). These TCs were propagated from epidermal cell suspensions after stimulation with TC activating agents (Con A, rIL-1, rIL-2), then evaluated for phenotypic features and TCR diversity. Similar to the in situ situation, most were CD4-/CD8+/TCR-alpha/beta+. In addition, two cultures contained TCR-gamma/delta+ cells; one of these determined to be an adherent CD4-/CD8+ population. Epidermal TCs were significantly (p less than 0.0001) more abundant in the sole than in the other body regions examined (i.e., 40 vs. 7 CD3+ cells/linear centimeter of epidermis) and seemed to have a particular affinity for the acrosyringial epithelium of eccrine sweat ducts. Moreover, the sole usually contained a greater number of CD8+ relative to CD4+ TCs, whereas the epidermal CD4/CD8 ratio in the trunk and extremities was quite variable, although the trend also was towards a slightly larger percentage of CD8+ cells. Collectively, our data suggest that the volar epidermis has a unique microenvironment which is responsible for both the higher density of TCs, preferentially CD8+, and lower number of LCs. This study has not only provided evidence for significant regional variability in the human epidermal TC population of normal skin, but also strengthens the concept for skin-associated lymphoid tissues (SALT), whereby memory TCs recirculate back to the epidermis and interact with resident antigen-presenting cells (i.e., LC).

Antibodies, Monoclonal↗

PubMed1990

Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.

Animals↗

PubMed1990

The implications of subunit interactions for the structure of the T cell receptor-CD3 complex.

Cell surface-expressed receptors are often multichain complexes. One of these, the T cell receptor (TcR) alpha/beta-CD3 complex, is known to contain at least seven chains: the alpha and beta TcR chains plus the gamma, delta, epsilon and two zeta chains from the CD3 complex (alpha beta gamma delta epsilon zeta 2). To gain insight into the structure of the complex we have used anti-peptide antisera specific for the individual subunits of the complex, and nonionic and ionic detergents to determine subunit interactions within the complex. Four closely associated pairs of chains could be identified: alpha beta, zeta 2, gamma epsilon and delta epsilon. Interactions between the TcR alpha beta and either gamma epsilon or delta epsilon could be observed in the apparent absence of other CD3 chains. Furthermore, a hierarchy in the strength of the association between the TcR and the individual CD3 chains could be distinguished: TcR epsilon greater than TcR delta greater than TcR gamma. The zeta 2 dimer could only be detected in "intact" TcR-CD3 complexes shedding no light on possible interactions with either the TcR or CD3-gamma, delta and epsilon chains. Finally, cross-linking experiments suggest a close spatial relationship between the TcR alpha beta and both the CD3-gamma and CD3-epsilon chains. The results demonstrate that the methods used give valuable information on subunit interactions in a cell surface-expressed receptor complex and suggest a TcR-CD3 complex in which two epsilon chains are present, one linked to gamma and the other to delta. The data further indicate that gamma epsilon and delta epsilon complexes interact directly with the TcR chains. Based on the observations a model for the structure of the TcR-CD3 is presented and discussed.

Amino Acid Sequence↗

PubMed1990

Structural comparison of alpha/beta and gamma/delta T cell receptor-CD3 complexes reveals identical subunit interactions but distinct cross-linking patterns of T cell receptor chains.

Here we compare the subunit interactions within the alpha/beta and gamma/delta T cell receptor-CD3 (TcR-CD3) complexes. By using different detergent solubilization protocols, subunit interactions can be defined within such cell surface receptor complexes. It was found that in both TcR-CD3 complexes analogous subunit interactions can be defined: CD3 gamma and CD3 delta each form a stable complex with separate CD3 epsilon chains. Both the TcR gamma/delta and TcR alpha/beta form stable receptor complexes with these CD3 gamma/epsilon and CD3 delta/epsilon subunit. A zeta homodimer is present in these TcR-CD3 complexes. However, biochemical cross-linking results in a covalent bond between the CD3 gamma chain and the TcR beta chain in alpha/beta TcR-CD3 complexes and between the CD3 gamma chain and the TcR delta in gamma/delta TcR-CD3 complexes. This latter observation was independent of the type of TcR gamma/delta studied (e.g. disulfide or non-disulfide linked). These cross-linking results either indicate that the TcR beta and delta chains are each others structural homolog or that in TcR-CD3 complexes both TcR chains (alpha and beta, gamma and delta) are spatially closely associated with the CD3 gamma chain. The identical subunit interactions in the alpha/beta and gamma/delta TcR-CD3 complexes indicate no large structural differences between these receptor complexes but rather suggest a striking conservation of crucial interaction between subunits in the TcR-CD3 complexes.

Animals↗

PubMed1990

Polymorphisms and diversity of T-cell receptor-gamma proteins expressed in mouse spleen.

Bulk populations of T-cell receptor (Tcr) gamma delta-expressing splenocytes from different inbred strains of mice were examined for the diversity of Tcr gamma delta proteins. Immunoprecipitations with anti-C gamma 1/2, anti-C gamma 4, and anti-V gamma 1 sera demonstrated that splenocytes from B10.BR, C57BL/6, and C57L strains of mice expressed the same array of Tcr gamma proteins, namely V gamma 1-C gamma 2, V gamma 1-C gamma 4, and V gamma 2-C gamma 1, although the Tcr gamma delta heterodimers observed for each of these strains were biochemically distinct. Examination of bulk splenic Tcr gamma delta heterodimers from several other inbred strains of mice demonstrated that each of the strains could be categorized into one of three basic phenotypes. For several reasons, the differences observed between the strains appeared to be solely dependent on polymorphisms of the Tcrg loci. First, F1 mice co-expressed both parental Tcr gamma delta phenotypes. Second, the distinguishing polymorphism between mice of phenotype 1 and phenotypes 2 or 3 was due to the presence of an N-linked glycosylation site within the Tcrg-Cl gene segment, previously described for BALB.B and C57BL/6 Tcrg-Cl genes. Finally, the V gamma 1-C gamma 4 polymorphism between mice of phenotype 3 and phenotypes 1 or 2 was due to differences in core protein size. Furthermore, the three defined Tcr gamma chains were expressed independently of the major histocompatibility complex (MHC) haplotype. Although no striking qualitative differences in Tcr gamma delta heterodimers were observed between strains (including those with autoimmune disorders), a quantitative difference in the relative amount of C gamma 4-encoded proteins was observed on Tcr gamma delta splenocytes from both newborn euthymic and adult athymic mice when compared to adult Tcr gamma delta splenocytes from euthymic mice. These results demonstrate that genetic polymorphisms exist among different mouse strains and suggest that selective developmental pressures may govern Tcr gamma delta expression.

Animals↗

PubMed1990

HLA-B37 and HLA-A2.1 molecules bind largely nonoverlapping sets of peptides.

T-cell recognition of peptides that are bound and presented by class I major histocompatibility complex molecules is highly specific. At present it is unclear what role class I peptide binding plays relative to T-cell receptor specificity in determination of immune recognition. A previous study from our group demonstrated that the HLA-A2.1 molecule could bind to 25% of the members of a panel of unrelated synthetic peptides as assessed by a functional peptide competition assay. To determine the peptide-binding specificity of another HLA class I molecule, we have examined the capacity of this panel of peptides to compete for the presentation of influenza virus nucleoprotein peptide NP-(335-350) by HLA-B37 to NP-peptide-specific HLA-B37-restricted cytotoxic T-lymphocyte lines. Forty-two percent of peptides tested were capable of inhibiting NP-(335-350) presentation by HLA-B37. Remarkably, none of these HLA-B37-binding peptides belong to the subset that was previously shown to bind to the HLA-A2.1 molecule. Only the NP-(335-350) peptide was capable of binding to both HLA-A2.1 and HLA-B37. These findings demonstrate that the peptide-binding specificities of HLA-B37 and HLA-A2.1 are largely nonoverlapping and suggest that, from the universe of peptides, individual HLA class I molecules can bind to clearly distinct subsets of these peptides.

Adult↗

PubMed1990

The kinetics of peptide binding to HLA-A2 and the conformation of the peptide-A2 complex can be determined by amino acid side chains on the floor of the peptide binding groove.

The ability of amino acid side chains in the floor of the peptide binding groove of HLA-A2 to affect the presentation of a viral peptide to peptide-specific cytotoxic T lymphocytes (CTL) has been examined. HLA-A2 molecules with naturally occurring single amino acid substitutions of Phe to Tyr at position 9 (HLA-A2.4a, Tyr9) and Tyr to Cys at position 99 (HLA-A2.4b, Cys99) and a site directed mutant with a Val to Leu substitution at position 95 (Leu95) were examined for their ability to present the influenza virus matrix M1 55-73 peptide and several sequence variants of the M1 peptide to a panel of 36 M1 55-73-specific HLA-A2.1-restricted CTL lines. The Leu95 molecule demonstrated enhanced kinetics of M1 peptide presentation and the ability to be sensitized by lower concentrations of the M1 peptide than the A2.1 molecule. The Tyr9 and Cys99 molecules exposed to M1 peptide were not recognized by 33 out of 36 CTL lines. The Tyr9 and Cys99 HLA-A2 molecules could bind the M1 55-73 peptide because at least one CTL line was found that could recognize each of these molecules that were exposed to the M1 peptide. CTL recognition patterns of variant M1 peptides presented by the Tyr9 molecule demonstrated that the amino acid at position 9 can be a critical determinant of the conformation of the peptide-A2 complex, and indicated that a particular peptide can bind in the HLA-A2 peptide binding groove in more than one conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Isolation and characterization of TGF-beta 2 and TGF-beta 5 from medium conditioned by Xenopus XTC cells.

TGF-beta 2 and -beta 5 have been purified from medium conditioned by Xenopus cultured cells (XTC) and identified based on their N-terminal amino acid sequence analysis and biological activity. When applied in high concentrations, Xenopus TGF-beta 2, like porcine TGF-beta 2, induces expression of mesodermal markers from cultured Xenopus ectodermal explants, whereas TGF-beta 5 is inactive in this assay. However, the TGF-beta 's could be separated from the major mesoderm-inducing activity present in XTC medium. Xenopus TGF-beta 2 and -beta 5 are approximately equivalent to TGF-beta 1 in their abilities to inhibit the growth of mink lung CCL-64 cells, induce anchorage-independent growth of rat NRK cells, inhibit the proliferation and antibody secretion of human B-lymphocytes, and stimulate chemotaxis of human monocytes. These data establish the functional activity of TGF-beta 5 and suggest that more complex multicellular systems, in contrast to most isolated cells, discriminate between the different TGF-beta s.

Amino Acid Sequence↗

PubMed1989

Identification of distinct T cell receptor (TCR)-gamma delta heterodimers using an anti-TCR-gamma variable region serum.

T cell hybridomas were generated from CD3+, CD4-, CD8- splenocytes and fetal thymocytes. V gamma 1-expressing proteins present on these murine TCR-gamma delta hybridomas were identified by using an anti-TCR V gamma 1 peptide serum. This antiserum specifically immunoprecipitated 41-kDa TCR V gamma-C gamma 4 chains and 31-kDa TCR V gamma-C gamma 1/2 chains from distinct heterodimers expressed on the TCR-gamma delta T cell hybridomas. The RNA from a hybridoma with a 31-kDa TCR-gamma chain hybridized with a V gamma 1 probe but failed to hybridize with a V gamma 2 probe. In contrast, the RNA from a hybridoma with a 32-kDa TCR-gamma chain hybridized with a V gamma 2 probe. This 32-kDa TCR-gamma chain was not immunoprecipitated by the anti-V gamma 1 serum. These data were consistent with the conclusion that the 31-kDa protein was the product of a V gamma 1 to C gamma 2 rearrangement, whereas the 32-kDa protein was the product of a V gamma 2 to C gamma 1 rearrangement. Furthermore, Southern analyses confirmed that the 32-kDa protein was the product of a V gamma 1.2-J gamma 2 rearrangement, and all three of the 41-kDa TCR-gamma chains were the results of V gamma 1.1-J gamma 4 rearrangements. This was the first demonstration at the clonal level of TCR-gamma proteins which use members of the V gamma 1 gene family, as well as the C gamma 2 constant region. Additional biochemical analyses of the TCR-gamma and -delta proteins from three independently derived C gamma 4-bearing T cell hybridomas suggested that most of the molecular mass diversity observed in the bulk subpopulation of peripheral C gamma 4-containing heterodimers may be contributed by the TCR-delta chains.

Amino Acid Sequence↗

PubMed1989

Specificity of peptide binding by the HLA-A2.1 molecule.

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.

Amino Acid Sequence↗

PubMed1989

Differential effects of amino acid substitutions in the beta-sheet floor and alpha-2 helix of HLA-A2 on recognition by alloreactive viral peptide-specific cytotoxic T lymphocytes.

Crystallographic studies of the HLA-A2 molecule have led to the assignment of a putative peptide binding site that consists of a groove with a beta-pleated sheet floor bordered by two alpha-helices. A CTL-defined variant of HLA-A2, termed HLA-A2.2F, differs from the common A2.1 molecule by three amino acids: a Leu to Trp substitution at position 156 in the alpha-2 helix, a Val to Leu substitution at position 95 in the beta-sheet floor of the groove, and a Gln to Arg substitution at position 43 in a loop outside of the groove. Another HLA-A2 variant, termed CLA, has a single Phe to Tyr substitution at position 9 that is sterically located adjacent to position 95 in the beta-sheet floor of the groove. We have determined which of the amino acid substitutions at positions 9, 43, 95, or 156 could individually affect recognition by panels of A2.1 allospecific and A2.1-restricted influenza viral matrix peptide-specific CTL lines, using a panel of site-directed mutants and CLA. Recognition by allospecific CTL lines was generally unaffected by any one of the amino acid substitutions, but was eliminated by the double substitution at positions 95 and 156. Allorecognition by some CTL lines was eliminated by a single substitution at position 9 or 95. In contrast, recognition by A2.1-restricted matrix peptide specific CTL was totally eliminated by a single substitution at position 9 or 156. The substitution at position 43 in a loop away from the peptide binding groove had no effect on allorecognition or matrix peptide recognition. These results indicate that amino acid residues in the floor or alpha-2 helical wall of the peptide binding groove of the HLA-A2 molecule can differentially affect allorecognition and viral peptide recognition.

Amino Acid Sequence↗

PubMed1989

A single amino acid substitution in HLA-A2 can alter the selection of the cytotoxic T lymphocyte repertoire that responds to influenza virus matrix peptide 55-73.

Previous studies have demonstrated that certain amino acid substitutions in the alpha two domain at positions 152 and 156 in the alpha two helix of the HLA-A2 molecule can affect presentation of the influenza virus matrix peptide M1 55-73 without abolishing binding of the M1 peptide. HLA-A2.1-restricted M1 55-73 peptide-specific CTL lines obtained from almost all HLA-A2.1+ individuals fail to recognize the M1 peptide presented by site-directed mutants of HLA-A2 that have either a Val----Ala or Val----Gln substitution at position 152 or a Leu----Trp substitution at position 156. Only one HLA-A2+ individual (donor Q66, HLA-A2,-B53,-B63) has been found who is able to generate a unique repertoire of HLA-A2-restricted M1 peptide-specific CTL that can recognize peptide presented by HLA-A2 mutants with either an Ala or Gln substitution at position 152 or a Trp substitution at position 156. These Q66 M1 peptide-specific CTL could be selected by stimulation with M1 peptide-pulsed transfectants that express the mutant HLA-A2 gene with the Trp substitution at 156. To determine if the presence of the unique CTL repertoire could be attributed to a variant HLA-A2 molecule in Q66, sequences were determined from polymerase chain reaction-amplified segments of the HLA-A2 RNA. Two different HLA-A2 genes were found expressed in Q66 cells: one is identical to HLA-A2.1 and the other is identical to HLA-A2.2F (Gln----Arg at position 43, Val----Leu at position 95, and Leu----Trp at position 156). These results demonstrate that a different CTL repertoire specific for HLA-A2 plus the M1 55-73 peptide is generated in an individual that expresses both HLA-A2.1 and HLA-A2.2F compared to individuals who express HLA-A2.1 alone, and that the unique repertoire can be selected by the presence of an HLA-A2 molecule with a single amino acid substitution at position 156.

Adult↗

PubMed1989

Murine T cells express a cell surface receptor for multiple extracellular matrix proteins. Identification and characterization with monoclonal antibodies.

Cell-cell and cell-extracellular (ECM) protein interactions are mediated through heterodimers termed integrins. We have demonstrated that dendritic epidermal T cell (DETC) lines adhere to the ECM proteins, fibronectin, fibrinogen, and vitronectin but not to collagen, laminin, or control proteins. This adhesion was blocked by the tetrapeptide RGDS, but not the control peptide, RGES. We have derived a hamster mAb H9.2B8, and a rat mAb, 8.18E12, from immunizations with DETC lines. The mAbs in combination, but not individually, specifically inhibited the adhesion of DETC lines to fibronectin, fibrinogen, and vitronectin. Immunoprecipitation analysis revealed that both mAbs reacted with a heterodimer composed of noncovalently linked 140- and 95-kD subunits. The 140-kD subunit can be reduced to 120- and 23-kD fragments. Although the two mAbs did not cross-compete for binding to DETC, sequential immunoprecipitation studies indicated that they react with the same 120-kD fragment. While all DETC cell lines and several T cell clones were reactive with the mAbs, the mAbs were not reactive with normal spleen, lymph node, thymus, or skin. Stimulation of splenic T cells with Con A or allogeneic cells induced mAb reactivity after 1 wk in vitro. These data demonstrate that a single lymphocyte receptor, with biochemical features characteristic of integrins, mediates RGD-dependent binding to the ECM proteins, fibronectin, fibrinogen, and vitronectin. Furthermore, since this integrin is expressed by long-term activated T cells, this receptor may play a physiological role in T cell function.

Animals↗

PubMed1989

Isolation of CD4- CD8- mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid.

The majority of peripheral T cells express a heterodimeric, alpha/beta T-cell receptor, which recognizes specific antigenic peptides bound to self major histocompatibility complex (MHC) molecules, and either the CD4 or CD8 surface markers. An additional subset of T cells, whose physiological function is unknown, express a distinct CD3-associated receptor composed of gamma and delta chains. This subset includes cells lacking both CD4 and CD8 surface markers, which may be involved in autoimmunity. The recognition specificity of the gamma/delta receptors is not well characterized and has been defined in only one case to date, a murine cell line which shows MHC-linked specificity. In this report, we describe the isolation of CD4- CD8-, gamma/delta TCR bearing T cell clones from the synovial fluid of a rheumatoid arthritis patient. These T cell clones respond specifically to mycobacterial antigens without MHC restriction.

Arthritis, Rheumatoid↗