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Biomedical subjects

L H Glimcher

Publications and source records attributed to L H Glimcher.

At least 163 records · Page 9Linked to original sources
PubMed1987

A single base mutation in an I-A alpha-chain gene alters T-cell recognition.

The interaction between the clonally selected T-cell antigen receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered I-Ak alpha-chain (Ak alpha) molecule has been examined in order to provide more information about the relationship between Ia structure and function. The cell line, 3J9, was derived from the TA3 B-cell hybridoma through a series of negative and positive immunoselection steps. The 3J9 mutant lacked the binding site recognized by the Ak alpha-specific monoclonal antibody 39J and failed to present antigen to two T-cell hybridomas out of a large panel of I-Ak-restricted T-cell hybridomas examined. Sequence analysis of the mutant Ak alpha gene showed a single base transition (G----A) that resulted in a glutamic acid to lysine substitution at amino acid 75 of the alpha 1 domain. This mutation confirms the importance of amino acid 75 in the expression of the Ia.19 epitope, demonstrates the involvement of this region in the presentation of antigen to specific T cells, and provides a further example of the multiple functional domains on the Ia molecule that are involved in antigen presentation.

Base Sequence↗
PubMed1987

Polyclonal B-cell activation by a B-cell differentiation factor B151-TRF2. IV. B151-TRF2-responsive F1 B cells consist of two separate populations capable of recognizing only one of the parental I-A products expressed on B cells.

Polyclonal IgM PFC responses of unstimulated B cells induced by a B cell differentiation factor, B151-TRF2, have been shown to involve an Ia-dependent process which is inhibitable by anti-Ia monoclonal antibody (mAb). Moreover, we have demonstrated that when T cell-depleted (B10 x B10.BR)F1 (H-2b/k) spleen cells are fractionated based on their ability to bind to a B10 (H-2b) monolayer, the B151-TRF2 responses of the adherent and nonadherent cell fractions are markedly inhibited by anti-I-Ab and anti-I-Ak mAbs, respectively. From these results, we hypothesized that B cell-recognition of self-I-A products expressed on B cells is involved in the B151-TRF2-induced polyclonal B cell activation. The present study examined the genetic and cellular requirements for the successful separation of F1 B cells with parental monolayers in order to prove recognition by B cells of self-I-A products expressed on B cells. The experiments utilizing monolayers from H-2 congenic strains revealed that identity at the I-A subregion of the H-2 complex between responding F1 B cells and monolayer cells was necessary and sufficient for the successful separation of F1 B cells. In support of this conclusion, purified B cells and B cell lines expressing only parental I-A products on the surface could function effectively as monolayers. Masking of I-A determinants expressed on the monolayer with anti-I-A mAb almost completely abolished the successful separation of F1 B cells, whereas pretreatment of Ia antigens on responding F1 B cells with anti-Ia mAbs did not affect the subsequent separation, indicating involvement of receptor-I-A product interaction rather than like-like interaction in the separation of F1 B cells by the monolayer. The B151-TRF2 responses of purified B cells obtained from athymic nude mice were specifically inhibited by the relevant anti-I-A mAb but not by anti-L3T4 and anti-LFA-1 mAbs, both of which were inhibitory to the Ia-restricted T cell responses. In addition, anti-Thy1.2 mAb plus complement-treated spleen cells from athymic F1 nude mice were successfully fractionated with parental monolayer into two separate populations with restriction specificity for only one of parental I-A products. These results negate the involvement of Ia-restricted T cells in the Ia-dependent process of the B cell activation. Finally, it was demonstrated that there was no apparent difference in the expression of respective parental I-A products between F1 B cell subpopulations adherent and nonadherent to parental monolayers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Regulation of class II gene expression: analysis in B cell stimulatory factor 1-inducible murine pre-B cell lines.

We have analyzed the mechanism of action of BSF-1 on class II gene expression in two murine pre-B cell lines that are at different stages of differentiation. In vitro transcription assays demonstrated that BSF-1 induced rapid transcription of class II genes within 1 hr by a mechanism that was independent of protein synthesis. Although cell surface expression of class II was detectable by 2 hr after induction and persisted for up to 96 hr after removal of BSF-1, levels of class II-specific mRNA and density of cell surface class II increased to a maximum between 24 and 72 hr after induction. Furthermore, prostaglandin E2 prevented the induction of class II gene expression by BSF-1. The effects of BSF-1 are unlikely to be mediated through calcium fluxes or activation of protein kinase C, since treatment with agents such as PMA and the calcium ionophore A23187 did not result in expression of class II molecules. Other lymphokines, such as IFN-gamma, IL 1, IL 2, and IL 3, did not affect class II gene expression in these two cell lines. These data provide further insight into the mechanisms by which class II gene expression is regulated in B cells.

Animals↗
PubMed1986

Ek beta mutant antigen-presenting cell lines expressing altered Ak alpha molecules.

Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites.

Animals↗
PubMed1986

Three distinct signals can induce class II gene expression in a murine pre-B-cell line.

Expression of class II genes of the major histocompatibility complex (MHC) has been studied in an Abelson-murine-leukemia-virus-transformed pre-B-cell line, R8, and its class II molecule (Ia)-negative variant, R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of a trans-acting factor(s) that can regulate class II gene expression. Furthermore, the T-cell-derived lymphokine B-cell-stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.

Abelson murine leukemia virus↗
PubMed1986

Identification of functional regions on the I-Ab molecule by site-directed mutagenesis.

Functional analysis of mutant class II major histocompatibility complex molecules has begun to identify regions important for antibody binding and for T-cell activation. By using in vitro mutagenesis directed at the beta 1 domain of the Ab beta gene we have constructed three structurally distinct mutant Ab beta genes. Each of these genes, as well as the wild-type Ab beta gene, was cotransfected together with the wild-type Ab alpha gene into the Ia-negative B-lymphoma cell line M12.C3. Transfection resulted in the successful synthesis and cell surface expression of three mutant class II antigens that showed serological and functional alterations as compared to the I-Ab antigens from the M12.C3 cell transfected with the wild-type gene. The variable patterns of both I-Ab-specific monoclonal antibody binding and activation of I-Ab-specific T-cell hybridomas show that the mutations result in the loss of structural epitopes required for both monoclonal antibody binding and for T-cell recognition. The data suggest that there are multiple sites on a single Ia molecule that are recognized by T helper cells and also that the tertiary conformation of the Ia molecule can be critical in the formation of such sites.

Animals↗
PubMed1986

Regulation of class II gene expression in a murine pre-B cell line.

Class II major histocompatibility complex (MHC) gene expression has been studied in an Abelson virus-transformed pre-B cell line R8, and its Ia-negative variant R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of trans-acting factors that can regulate class II gene expression. Furthermore, the T cell-derived lymphokine B cell stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.

Animals↗
PubMed1985

Direct evidence that a class II molecule and a simple globular protein generate multiple determinants.

We have examined the individual contributions of the I-A kappa alpha chain, the I-A kappa beta chain, and the foreign antigen hen egg-white lysozyme (HEL) in the formation of the determinant being recognized by the T cell receptor. As functional probes we have used (a) a panel of 10 HEL-specific T cell hybridomas, (b) a panel of antigen-presenting cells (APC) possessing mutations in either the I-A kappa alpha or I-A kappa beta chains, and (c) proteolytic fragment of HEL and related synthetic peptides. The ability of the I-A kappa beta and I-A kappa alpha mutant cell lines to present antigen to the 10 T cell hybridomas divided these T cells into six distinct groups. These HEL-specific T cells therefore appear to recognize several distinct domains on the I-A kappa molecule. The 10 T cell hybrids were then shown to recognize at least three distinct determinants on the HEL molecule, with 8 of the 10 hybrids recognizing one of two major determinants HEL(46-61) or HEL(34-45). Combining the response patterns to the panel of I-A kappa mutant APC lines with the antigen specificity revealed that the 10 T cell hybrids recognized at least eight unique determinants formed by the I-A kappa alpha chains, I-A kappa beta chains, and HEL peptides. This analysis provides direct evidence that a large number of different determinants or T cell receptor ligands can be generated from a single Ia molecule and a simple globular protein.

Animals↗
PubMed1985

Both L3T4+ and Lyt-2+ helper T cells initiate cytotoxic T lymphocyte responses against allogenic major histocompatibility antigens but not against trinitrophenyl-modified self.

This study characterizes the T helper (Th) cells that initiate primary cytotoxic T lymphocyte (CTL) responses against allogeneic and trinitrophenyl (TNP)-modified self class I major histocompatibility (MHC) determinants. We show that two distinct Th cell subsets participate in allospecific CTL responses: (a) an L3T4+,Lyt-2- class II-restricted Th cell population, and (b) an L3T4-,Lyt-2+ class I-restricted Th cell population. Both of these T cell subpopulations were shown to function in allospecific CTL responses as helper cells by their ability to show synergy with allospecific CTL precursors. Thus, primary class I allospecific CTL responses represent an immune response involving not only L3T4+ Th cells, but Lyt-2+ Th cells as well. One of the necessary functions performed by both L3T4+ and Lyt-2+ Th cell populations in allospecific CTL responses was found to be the secretion of interleukin 2. Finally, despite the many similarities between anti-allo- and anti-TNP-CTL responses, anti-TNP-CTL responses were found to be mediated by only L3T4+ Th cells, not by Lyt-2+ Th cells. Consequently, Lyt-2+ Th cells appear to be a helper cell population that is primarily involved in MHC-specific immune responses.

Animals↗
PubMed1985

An Ia-positive mouse T-cell clone is functional in presenting antigen to other T cells.

In this report we present data demonstrating the endogenous expression of I-region associated (Ia) antigens on a cloned line of mouse T cells, CTLL, as well as transcription of the invariant chain gene in these cells. We demonstrate further that this Ia-bearing T-cell clone, CTLL, can utilize the expressed Ia molecules to present antigen to Ia-restricted antigen-specific T cells.

Animals↗
PubMed1985

Analysis of responder cell-cell interactions in the syngeneic mixed leukocyte response.

In a previous study we identified the target antigens on stimulator cells in the murine syngeneic mixed leukocyte response (MLR) as self Ia molecules. The experiments reported here utilized analysis of log-log cell number-response titration slopes to study the responder T-cell population in the syngeneic MLR. The data indicated that in the peripheral lymphoid population at least two cell populations interact to produce a murine syngeneic MLR. One of these cell types appeared to be a member of an Lyt 2- subset, another a member of an Lyt 2+ subset. A potential third cell type, which was detected in the presence of (PMA)-induced EL4 supernatants, was found in the thymus, an organ that did not appear to contain either of the other two subsets. Experiments with adult thymectomized and neonatal mice suggested that the cell populations involved in the syngeneic MLR were mature T cells and were not an early differentiation state of T lymphocytes whose ability to be stimulated by Ia antigens alone was a transient event.

Animals↗
PubMed1985

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas.

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.

Animals↗
PubMed1985

Evidence that autoreactive T hybridomas recognize multiple epitopes on the I-Ak molecule.

In this study we attempt to define the number of determinants on the I-Ak molecule recognized by a panel of autoreactive I-Ak-specific T cell hybridomas. The recognition sites or histotopes of a panel of autoreactive I-Ak-restricted T cell hybridomas was determined in two ways: 1) by their ability to be activated by a panel of A betak and A alphak mutant antigen-presenting cell lines, and 2) by inhibition of activation by anti-I-Ak monoclonal antibodies. The results are most consistent with the presence of multiple distinct recognition sites on the I-Ak molecule. However, an alternative explanation, that the T cell hybridomas recognize the same self epitope on Class II molecules, but with different affinity, cannot be excluded.

Animals↗
PubMed1985

Prostaglandin E2 inhibits the activation of cloned T cell hybridomas.

To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.

Animals↗
PubMed1985

Complex regulation of class II gene expression: analysis with class II mutant cell lines.

Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma-irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I-Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I-Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide-induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells.

Animals↗
PubMed1985

DNA-mediated transfer of major histocompatibility class II I-Ab and I-Abm12 genes into B lymphoma cells: molecular and functional analysis of introduced antigens.

A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.

Animals↗