Biomedical subjects

J A Bluestone

Publications and source records attributed to J A Bluestone.

At least 217 records · Page 12Linked to original sources

PubMed1988

Cytotoxic T lymphocyte recognition of novel allodeterminants expressed on in vitro selected H-2Kb mutants.

In the present study, in vitro derived H-2Kb mutants have been examined by alloreactive CTL. Two mutants, R8.24 and R8.246, have been shown to express novel determinants detected by CTL generated against some but not all in vivo derived Kb mutants. BDF1 anti-bm3, anti-bm11, anti-bm19, anti-bm23, and anti-bm6 CTL populations lyse the two R8 variants. The novel determinants expressed on the R8 mutants detected by the bm3 and bm23-specific CTL appear to differ from the determinant recognized by the bm6-specific CTL. No new serologically defined determinants were detected on any of 18 independent R8 variants. However, these results do not rule out the existence of new determinants which could be recognized by antibodies. Finally, the relationship between the T cell recognition of the in vivo and in vitro derived mutants and their use in understanding the structure/function relationships between the immune response and class I Ag based on recent crystallographic analyses is discussed.

Animals↗

PubMed1988

Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor.

Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues.

Amino Acid Sequence↗

PubMed1988

Selective expression of the Ly-6C.2 epitope on Lyt-2+ T cells that can develop in the absence of thymic influence.

The present study focused on attempts to correlate the expression of T cell differentiation antigens and development. A series of mAb was developed that recognized an Ly-6.2-linked Ag, Ly-6C.2. These mAb reacted with a subset of peripheral Lyt-2+ T lymphocytes but not Lyt-2+ thymocytes. Functional analysis of the Lyt-2+, Ly-6C.2- and Lyt-2+, Ly-6C.2+ subsets demonstrated that both cell types responded in class I and class II allogeneic MLC as well as in virus-specific cell-mediated cytolysis assay, although precursor frequency analysis suggested that the majority of precursor CTL were Ly-6C.2+. The marker did not appear to represent an activation Ag because Ly-6C.2-, Lyt-2+ precursor CTL generated Ly-6C.2-, Lyt-2+ CTL. Finally, virtually all splenic Lyt-2+ T cells from athymic (nu/nu) mice were Ly-6C.2+ whereas all thymic CTL were Ly-6C.2-. These results suggested that Ly-6C.2+, Lyt-2+ T cells may represent a distinct lineage of Lyt-2+ T cells that can develop in the absence of thymic influence.

Animals↗

PubMed1988

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains.

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.

Animals↗

PubMed1988

Thymus-dependent and thymus-independent developmental pathways for peripheral T cell receptor-gamma delta-bearing lymphocytes.

To elucidate the developmental pathways of T cells that bear TCR gamma delta, we have analyzed the kinetics of expression and biochemical characteristics of gamma delta receptors in the thymus and spleen of normal and athymic (nude) mice, as well as nude mice engrafted with neonatal thymuses. TCR gamma delta-bearing thymocytes and splenocytes have a CD4-8- phenotype, and both populations express products of the C gamma 1 locus. TCR gamma delta-bearing cells develop in the thymus before their appearance in the spleen. Young nude mice have no detectable TCR gamma delta-bearing cells in their spleens. When young nude mice are given thymus grafts, TCR gamma delta-bearing cells of host origin first develop in the engrafted thymus, followed by their appearance in the spleen. In the absence of a thymus graft, the spleens of old nude mice eventually develop small numbers of TCR gamma delta + cells, as well as TCR alpha beta + cells. These results demonstrate that there is a major thymic-dependent pathway for TCR gamma delta expression, as well as a minor thymic-independent pathway seen in older nude mice. The development of TCR gamma delta + cells in the thymus before their appearance in the spleen, both in normal ontogeny as well as in the thymus-engrafted nude mouse model, suggests that thymic TCR gamma delta + cells are precursors of the thymus-dependent population of peripheral TCR gamma delta + cells.

Aging↗

PubMed1988

Effects of in vivo administration of anti-T3 monoclonal antibody on T cell function in mice. I. Immunosuppression of transplantation responses.

Anti-T3 mAb are being increasingly used clinically in the treatment of organ graft rejection. However, there has not previously been a murine model in which the effects of these mAb on the immune system could be studied in vivo. We have established such a model using the anti-murine-T3 mAb, 145-2C11. Administration of 145-2C11 led to rapid depletion of T cells from peripheral blood and suppression of skin graft rejection. However, depletion of T cells from spleen and lymph node was both delayed and incomplete. Full recovery of T cell number was dependent on the presence of a thymus, but treatment of thymectomized animals revealed that depletion was not the mechanism by which the mAb induced immunosuppression. Rather, alterations in TCR expression may play a more important role. TCR had modulated from T cells in spleen and lymph node early after treatment, and TCR expression remained subnormal for at least 51 days posttreatment. However, subnormal TCR expression alone could not fully explain the observed T cell dysfunction, inasmuch as a period of time existed after TCR re-expression during which T cells appeared to be anergic to CTL and MLR reactivity. These findings implicate T cell dysfunction as an important element in the induction of immunosuppression after anti-T3 administration.

Animals↗

PubMed1988

Proliferation and production of IL-2 and B cell stimulatory factor 1/IL-4 in early fetal thymocytes by activation through Thy-1 and CD3.

To examine which cell surface molecules can operate as transducers of activation signals to early fetal thymocytes, we analyzed the ability of mAb to CD3 and Thy-1 to induce fetal thymocyte activation. Both proliferation and lymphokine secretion were used as measures of activation. We show that anti-CD3 antibodies induce activation of fetal thymocytes as early as day 13 of fetal thymus development, 2 days before CD3 can be detected by flow cytometry. In addition, an alternative activation signal can be delivered to fetal thymocytes through the Thy-1 molecule as early as it is expressed, i.e., day 13. Both CD3- and Thy-1-mediated activation of day 15 fetal thymocytes results in expansion of cells expressing a CD3-gamma delta receptor complex; no CD3-alpha beta receptor complex could be detected. IL-2 production induced by CD3- and Thy-1-induced activation of fetal thymocytes is evident at the 13th day of gestation. Finally, an additional lymphokine B cell stimulatory factor-1 (BSF-1)/IL-4 (so far known only to be produced by mature CD3- cells), is also produced by fetal thymocytes. The results demonstrate that at least two cell surface molecules, Thy-1 and CD3, can function as pathways of activation in fetal thymocytes, and that at least two lymphokines, IL-2 and BSF-1/IL-4, are produced upon activation. These findings may well reflect a role for the early appearance of CD3- cells in thymus ontogeny.

Animals↗

PubMed1988

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules.

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.

Animals↗

PubMed1988

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones.

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.

Animals↗

PubMed1988

CD3-associated heterodimeric polypeptides on suppressor hybridomas define biologically active inhibitory cells.

We have investigated the relationship between CD3 expression and the suppressor T-cell function. We have isolated stable clonal cell lines of the F12.23 suppressor T-cell hybridoma that are either CD3+ or CD3-. These lines were subjected to functional assays including inhibition of in vivo hapten-specific delayed-type hypersensitivity responses, in vitro hapten-specific interleukin 2 responses, as well as hapten-specific cytotoxic T-lymphocyte assays. In all assays, the functional suppressor phenotype absolutely correlated with CD3 surface expression. Furthermore, we have immunoprecipitated heterodimeric proteins that share molecular features with some receptor polypeptides previously described. CD3 polypeptides found on the surface of suppressor T cells are phosphorylated after phorbol ester stimulation. Collectively these studies unambiguously define the suppressive supernatant function as a product of CD3+ receptor-bearing T cells.

Animals↗

PubMed1988

In vivo administration of anti-CD3 monoclonal antibody can activate immune responses thus preventing malignant tumor growth.

Anti-CD3 monoclonal antibodies (mAb) have been shown to suppress T cell-mediated immune responses both in vitro and in vivo. However, in vitro studies with these antibodies have also demonstrated that they possess potent mitogenic properties, raising the possibility that they might be capable of potentiating immune responses in vivo. In this regard, we have recently shown that an anti-CD3 mAb can activate murine T cells in vivo. Furthermore, low doses of antibody induce interleukin 2 (IL-2) receptor expression and enhanced proliferation to allogeneic major histocompatibility complex (MHC) antigen without detectable modulation or blocking of the T cell receptor and without suppression of T cell-mediated immune responses. In light of these findings, we investigated the ability of low dose anti-CD3 to enhance an anti-tumor response directed against the malignant murine UV-induced skin tumor, 1591-Pro-4L. Low dose anti-CD3 administration resulted in enhanced in vitro anti-tumor activity and prevented tumor outgrowth in approximately two-thirds of animals treated at the time of tumor inoculation. Furthermore, these animals displayed lasting tumor-specific immunity. These results suggest that anti-CD3 mAb can be utilized for the enhancement of anti-tumor responses in vivo and may have general application in the treatment of immunodeficiency.

Animals↗

PubMed1987

Role of accessory molecules in signal transduction of cytolytic T lymphocyte by anti-T cell receptor and anti-Ly-6.2C monoclonal antibodies.

Accessory molecules present on the cell surface of cytolytic T lymphocytes (CTL) play an important role in their activation. Antigen-specific recognition by CTL is inhibited by antibodies against Lyt-2, L3T4, or LFA-1 molecules. Presently it is not known whether these molecules function by binding a ligand such as class I or class II on the target cell or by delivering a signal that down-regulates T cell activation. In the present study we utilized anti-T cell antibodies including anti-T3 and anti-T cell receptor (alpha/beta) as well as an anti-Ly-6.2C monoclonal antibody to activate CTL clones to kill irrelevant targets or secrete BLT esterase. The redirected lysis assay system utilizes the fact that heteroconjugates between anti-T3, and anti-T cell receptor, or anti-Ly-6.2C and anti-trinitrophenyl can trigger CTL lysis of trinitrophenyl-coupled targets that did not express antigen. In this system anti-Lyt-2 antibodies as well as anti-LFA-1 antibodies inhibited triggering via T cell receptor-related molecules but not via the anti-Ly-6.2C heteroconjugate. In addition, the anti-Lyt-2 was shown to inhibit conjugate formation in the heteroaggregate assay system suggesting that the anti-Lyt-2 antibodies acted early in inhibiting CTL activity. Similar results were observed in a system in which the CTL clones were triggered to secrete a BLT-esterase-like activity in the absence of target cells. Anti-T3 coated on plastic was shown to activate BLT-esterase secretion. This secretion was inhibited by anti-Lyt-2 and anti-LFA-1. Thus, it would appear that both the Lyt-2 molecule and the LFA-1 molecule act as signal-transducing elements involved in CTL activation. In particular, the Lyt-2 molecule appears to preferentially function in receptor-mediated T cell activation.

Animals↗

PubMed1987

A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Amino Acids↗

PubMed1987

T cell receptor tyrosine phosphorylation. Variable coupling for different activating ligands.

We have previously reported (Samelson, L.E., Patel, M.D., Weissman, A.M., Harford, J.B., and Klausner, R.D. (1986) Cell 46, 1083-1090) that T cell activation by antigen is associated with activation of two biochemical pathways. In this scheme two protein kinases are activated by stimulation of the T cell antigen receptor (TCR). These kinases phosphorylate two different chains of the TCR complex. Protein kinase C is responsible for the phosphorylation of the gamma, and, to a lesser extent, the epsilon chains of the receptor on serine residues while the activation of an unidentified tyrosine kinase leads to phosphorylation of the p21 subunit of the receptor on tyrosine residues. In addition to activation by specific antigens, T cells can be functionally activated in vitro by the addition of antibodies that bind either the antigen receptor or the Thy-1 molecule, an entity independent of the receptor. We have used antibodies directed against these molecules and show that they result in the same dual kinase activation observed with antigen stimulation. In addition we have compared the three ligands, antigen, and antibodies directed against the epsilon chain of the TCR or against Thy-1, in terms of how they couple to the two kinase pathways. Activation of phosphatidylinositol breakdown and TCR phosphorylation on serine by all three stimuli are sensitive to cAMP inhibition. In contrast, only antigen-stimulated tyrosine kinase activation is sensitive to cAMP while the two antibody reagents activate the tyrosine kinase in a manner that is entirely insensitive to cAMP inhibition.

Animals↗

PubMed1987

Activation of murine T lymphocytes with monoclonal antibodies: detection on Lyt-2+ cells of an antigen not associated with the T cell receptor complex but involved in T cell activation.

A new T cell molecule defined by the mAb 143-4-2 has been identified that is involved in T cell activation. The expression of the 143-4-2-defined epitope is linked to the previously characterized Ly-6 locus and restricted to bone marrow cells and to a subset of peripheral Lyt-2+ cells. In comparison to other anti-Ly-6.2 mAb, the 143-4-2 mAb appears to be directed at an allogeneic determinant of the Ly-6.2C molecule. The anti-Ly-6.2C antibody can promote the lysis of antigen-non-bearing target cells by alloreactive CTL clones, and in the presence of cofactors (PMA or IL 2) induces a subset of Lyt-2+ cells to proliferate, perhaps through an autocrine pathway. Although the antibody described has antigen-like effects as described for anti-TcR complex reagents, studies performed with a recently derived anti-murine T3 mAb suggest that the Ly-6.2C molecule is not associated on the cell surface with components of the TcR complex. Nevertheless, cell surface expression of the TcR complex is required for optimal triggering of T cells via the Ly-6.2C molecule. Because Ly-6.2C determinants are expressed in bone marrow and not in the thymus, the possibility is considered that expression of this molecule identifies a distinct subset of extrathymically derived T cells.

Animals↗

PubMed1987

Production of a T cell hybridoma that expresses the T cell receptor gamma/delta heterodimer.

We have produced a T cell hybridoma line by fusion of an IL-2-dependent, long-term T cell receptor (TCR) gamma/delta+ Thy-1+, bone marrow-derived, dendritic epidermal cell line to the BW5147 tumor line. The resultant hybridoma was rapidly growing, lymphokine independent, and expressed T3 in association with the TCR gamma/delta heterodimer. Several subclones of the hybridoma line produced easily detectable levels of IL-2 after stimulation by anti-T3 or Con A. The availability of these cloned cell lines should greatly facilitate further functional, biochemical, and molecular studies of the TCR delta chain.

Animals↗

PubMed1987

Antigen receptor-triggered secretion of a trypsin-type esterase from cytotoxic T lymphocytes.

Exocytosis of cytolysin-containing granules from cytotoxic T lymphocytes (CTL) was studied with the use of granule enzyme (BLT esterase) as a convenient biochemical marker. Using cloned CTL, we demonstrate here that BLT esterase secretion into the supernatant is specifically triggered by antigen-bearing target cells and that this secretion is inhibited by soluble monoclonal antibodies against the antigen-specific T cell receptor (TcR). Immobilized anti-receptor antibodies induced efficient enzyme secretion in the absence of target cells, thus implying a direct involvement of TcR complex in triggering exocytosis of granules. These results support the role of the granule exocytosis in CTL functions and provide a quantitative and direct assay of a rapid CTL functional response to antigenic stimulation.

Animals↗

PubMed1987

Cytotoxic T lymphocyte recognition of a xenogeneic major histocompatibility complex antigen expressed in transgenic mice.

Introduction of a porcine major histocompatability complex (MHC) class I gene (PD1) into the genome of a C57BL/10 (B10) mouse has been shown to lead to cell surface expression of the porcine MHC antigen, SLAPD1 in a transgenic mouse. The PD1 product expressed on spleen cells from the transgenic mice stimulated B10 spleen cells in a mixed lymphocyte culture to generate PD1-specific cytotoxic T lymphocytes (CTL). The CTL were PD1 specific since they lysed transgenic splenic blast cells and PD1-transfected L cells, but not B10 blasts or control L cells. The CTL were L3T4-, Lyt-2+ and their activity was partially inhibited by either anti-Lyt-2 antibody or by anti-swine MHC alloantibodies. The repertoire of responding B10 anti-transgenic CTL was assessed by examining their cross-reactivity on a series of murine allogeneic targets. The B10 anti-transgenic CTL showed some cross-reactivity on conventional allogeneic targets, but reacted strongly on a series of mutant H-2Kbm blast cells. In addition, B10 anti-B6.cH-2bm6 CTL cross-reacted extensively on the transgenic target cells. These results demonstrated that normal B10 CTL possess a repertoire specific for the products of the xenogeneic class I gene PD1, that this repertoire is cross-reactive with the conventional alloreactive CTL repertoire, and that there exists an unanticipated relationship between PD1-specific CTL and CTL specific for Kb mutant determinants.

Animals↗