Biomedical subjects

B Benacerraf

Publications and source records attributed to B Benacerraf.

At least 19 recordsLinked to original sources

PubMed1992

Murine B7 antigen provides a sufficient costimulatory signal for antigen-specific and MHC-restricted T cell activation.

We have previously shown that the murine B7 (mB7) molecule, when expressed in Chinese hamster ovary cells in stable fashion, can costimulate with anti-CD3 mAb or Con A to induce T cell activation. We have now derived, by gene transfection, Chinese hamster ovary cell lines that express the I-Ad molecule, either alone or in context with mB7. We have analyzed these transfectants for their capacity to present Ag to murine CD4+ T lymphocytes. I-Ad/mB7-double transfectants were able to stimulate mixed lymphocyte reactions and to present peptide Ag to specific T cells. Chinese hamster ovary cells that expressed only the I-Ad molecule were not able to stimulate T cell proliferation in these systems. Thus, the mB7 protein is a sufficient costimulatory molecule for the physiologic, Ag-dependent/MHC-restricted activation of murine CD4+ T cells. Stimulation of T cell bulk cultures resulted predominantly in the production of IL-2 and not of IL-4. The costimulatory activity of mB7 is not, however, restricted to the IL-2-secreting subset. We have identified one IL-4-secreting T cell clone, CDC35, which is responsive to mB7 triggering. Finally, we present experiments that suggest that mB7 and peptide/MHC complexes need to be expressed on the same cell for optimal induction of T cell activation.

Animals↗

PubMed1992

Analysis of the association of peptides of optimal length to class I molecules on the surface of cells.

The association of major histocompatibility complex (MHC) class I molecules on the surface of cells with synthetic antigenic peptides of eight or nine amino acid residues was examined. Peptides were synthesized that correspond to the antigenic sequences from ovalbumin and influenza nucleoprotein believed to be naturally processed and presented by cells with Kb and Db MHC class I molecules, respectively. Consistent with the results of others, these peptides were 10(3)-10(5) times more active in stimulating specific T cells as compared to peptides of longer sequences. When cells are incubated with these peptides at less than 0.01-0.1 microM, the association of the peptides with class I molecules is dependent on (i) the reassociation of free beta 2-microglobulin from the extracellular fluids, (ii) a process that requires cells to be metabolically active, or (iii) stabilization of class I heterodimers by chemical crosslinking. In contrast, when cells are incubated with these peptides at greater than 0.1-1.0 microM, the peptides associate with class I molecules in the absence of exogenous beta 2-microglobulin, energy, or chemical crosslinking. Antigen competition experiments suggest that the class I molecules that bind peptides offered at high concentration become only transiently receptive to binding peptide. The concentration of peptides required for presentation to T cells under these conditions corresponds to those that stabilize Kb molecules on the surface of RMA-S mutant cells in the absence of exogenous beta 2-microglobulin. These results support the concept that the receptivity of class I molecules on cells is determined by the dissociation of beta 2-microglobulin from MHC class I that lacks bound peptides.

Animals↗

PubMed1992

Heterogeneity in antigen processing by different types of antigen-presenting cells. Effect of cell culture on antigen processing ability.

The ability of normal B cells, peritoneal macrophages, and splenic APC to process and present OVA to a panel of T-T hybridomas with different specificities was investigated. In all cases, B cells were less efficient than unfractionated splenocytes in presenting OVA or its peptides. However, when the presentation of native Ag was compared to the presentation of peptides, it was obvious that there were marked differences in the ability of these two APC populations to generate different epitopes from OVA. Leupeptin inhibits the processing of selected epitopes from native OVA differently when it was presented by spleen cells or B cells, suggesting that these two APC populations differ in their protease content. The effect of in vitro culture on the ability of splenic and peritoneal APC to process OVA was also investigated. Native OVA presentation by macrophages and spleen cells was affected by in vitro culture, more for some epitopes than for other epitopes. In contrast, presentation of exogenous peptides by paraformaldehyde-fixed APC was either not affected by previous culturing for 3 days, or very much improved. Altogether, these data demonstrate that different epitopes on the same protein may be independently and differentially processed by B cells and spleen cells. Furthermore, the precise peptides that are produced may vary with the physiologic state of the APC.

Amino Acid Sequence↗

PubMed1992

Serum proteases alter the antigenicity of peptides presented by class I major histocompatibility complex molecules.

Any effect of serum on the antigenicity of peptides is potentially relevant to their use as immunogens in vivo. Here we demonstrate that serum contains distinct proteases that can increase or decrease the antigenicity of peptides. By using a functional assay, we show that a serum component other than beta 2-microglobulin enhances the presentation of ovalbumin peptides produced by cyanogen bromide cleavage. Three features of this serum activity implicate proteolysis: it is temperature dependent, it results in increased antigenicity in a low molecular weight peptide fraction, and it is inhibited by the protease inhibitor leupeptin. Conversely, presentation of the synthetic peptide OVA-(257-264) is inhibited by serum. This inhibition is unaffected by leupeptin but is blocked by bestatin, a protease inhibitor with distinct substrate specificities. Implications for peptide-based vaccine design and immunotherapy are discussed.

Animals↗

PubMed1992

Diversity in MHC class II ovalbumin T cell epitopes generated by distinct proteases.

It is generally accepted that a limited number of T cell epitopes are generated by APC from an immunogenic protein. To ascertain the number of determinants on OVA recognized in the context of the H-2s haplotype, we generated 19 T-T hybridomas against OVA and H-2s and we synthesized 46 overlapping peptides spanning the entire protein. Eighteen T-T hybrids were stimulated by eight different peptides. The peptide recognized by one T cell hybrid was not identified. The effect of four protease inhibitors on the processing and presentation of OVA by the LS.102.9 B cell hybridoma seemed to implicate several groups of proteases in the processing of this Ag. Alkylation of cysteine residues with iodoacetic acid showed in a few cases a dramatic decrease in the capacity of OVA to stimulate T-T hybrids recognizing cysteine-free peptides. In contrast, two T-T hybrids recognizing cysteine containing peptides were not affected by the alkylation, suggesting that alkylation inhibited the processing of OVA without affecting peptide interaction with class II MHC molecules. These data demonstrate that the repertoire of peptides generated by APC from OVA is not limited to one or few immunodominant peptides, and results from the activity of several endopeptidases and/or exopeptidases. In addition, the structure of the Ag (native or denatured) was shown to affect the efficiency with which different epitopes are generated.

Animals↗

PubMed1992

Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells.

The murine B7 (mB7) protein is a potent costimulatory molecule for the T-cell receptor (TCR)-mediated activation of murine CD4+ T cells. We have previously shown that stable mB7-transfected Chinese hamster ovary (CHO) cells but not vector-transfected controls synergize with either anti-CD3 monoclonal antibody-induced or concanavalin A-induced T-cell activation, resulting ultimately in lymphokine production and proliferation. We now have generated a hamster anti-mB7 monoclonal antibody. This reagent recognizes a protein with an apparent molecular mass of 50-60 kDa. The mB7 antigen is expressed on activated B cells and on peritoneal exudate cells (PECs). Antibody blocking experiments demonstrate that mB7 is the major costimulatory molecule expressed by PECs for the activation of murine CD4+ T cells. This suggests an important role for mB7 during immune-cell interactions. We have also surveyed a panel of murine cell lines capable of providing costimulatory activity. Our results indicate that mB7 is the major costimulatory molecule on some but not all cell lines and that there may be additional molecules besides mB7 that can costimulate the activation of murine CD4+ T cells.

Animals↗

PubMed1992

Chemical cross-linking of class I molecules on cells creates receptive peptide binding sites.

Class I heterodimers on the surface of cells are generally unreceptive to binding peptides in the absence of exogenous beta 2-microglobulin. Paraformaldehyde covalently cross-links beta 2-microglobulin to class I heavy chains in situ and stabilizes empty class I heterodimers. Functionally, this cross-linking creates receptive class I peptide binding sites by acting on beta 2-microglobulin-associated molecules. The presentation of preexisting peptide-class I complexes is also enhanced. These findings support a model whereby a structural alteration, the dissociation of beta 2-microglobulin, limits the existence of receptive class I molecules on normal cells and may control the half-life of active class I molecules.

Animals↗

PubMed1992

The class II MHC-restricted presentation of endogenously synthesized ovalbumin displays clonal variation, requires endosomal/lysosomal processing, and is up-regulated by heat shock.

LB27.4 cells (a B lymphoblastoid APC) were transfected with a plasmid containing an OVA cDNA. Functional analysis of six independent clones yielded three patterns of MHC-restricted presentation of the endogenously synthesized OVA. A clone displayed either: 1) strong class I and class II-restricted presentation, 2) strong class I but little or no class II-restricted presentation or, 3) only a modest class I-restricted presentation. There was no clonal variation in class II-restricted presentation of exogenous Ag or in the amount of surface class I or II molecules. Heat shock increased the presentation of endogenous but not exogenous Ag with class II. These results indicate that an endogenously synthesized Ag both constitutively and during heat shock can gain access to the class II, MHC-restricted, presentation pathway. The amount of OVA synthesized by a cell correlated with whether OVA-class II complexes were detected. However, the amount of OVA secreted into the extracellular fluid was not sufficient to sensitize APC, which suggests that endogenously synthesized OVA enters the class II pathway of Ag presentation by an intracellular route rather than by an extracellular/reuptake route. Also, the functional and quantitative analysis of the clones suggests that endogenously synthesized OVA was presented more efficiently with class I as compared to class II-MHC molecules. Leupeptin and chloroquine inhibited the class II-restricted presentation of endogenously synthesized OVA. Together these results indicate that endogenously synthesized OVA can gain access to an endosomal/lysosomal compartment via an intracellular route and be processed and presented in association with class II-MHC molecules.

Animals↗

PubMed1992

Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.

Animals↗

PubMed1991

The generation of immunogenic peptides can be selectively increased or decreased by proteolytic enzyme inhibitors.

The ability of splenic APC and a B cell hybridoma (LS.102.9) to process and present OVA to a panel of T-T hybridomas with different fine specificities was investigated. Splenic APC process and present OVA to all the T-T hybrids. The B cell hybridoma could similarly process and present OVA to some T-T hybrids but was very inefficient in stimulating two of the T cell hybridomas. The presentation of native OVA to these two T-T hybrids was significantly increased by leupeptin. Pulsing experiments demonstrated that leupeptin acted on the APC at a step before the processed Ag was displayed on the cell surface in association with MHC molecules. Leupeptin has no effect on the presentation of OVA peptides by LS.102.9 to the T-T hybrids. Leupeptin inhibits the generation of the epitopes of OVA that LS.102.9 produces under basal conditions. We also surveyed the effect of other protease inhibitors and observed similar augmenting and inhibitory effects on the presentation of selected OVA epitopes. The augmentation of processing by a protease inhibitor indicates that in the lysosomal/endosomal compartment proteases have capacity to both generate and destroy immunogenic peptides. Our data suggest that protease inhibitors could potentially be used as immunomodulators and are discussed in terms of physiology of the lysosomal/endosomal compartment.

Animals↗

PubMed1991

Dissociation of beta 2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface.

A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide-binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to binding peptides yet readily associate with peptides in the presence of exogenous beta 2-microglobulin. These results have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved.

Animals↗

PubMed1991

Low temperature and peptides favor the formation of class I heterodimers on RMA-S cells at the cell surface.

RMA-S murine cells have a mutation that interferes with the assembly of class I major histocompatibility complex (MHC) heterodimers and are deficient in the expression of class I molecules on the cell surface. The mutant phenotype has been reported to be normalized upon incubation of RMA-S cells at 25 degrees C. We find that much of the increased expression of class I heterodimers is dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2-microglobulin. Furthermore, epitopes that are associated with class I MHC molecules that have bound xenogeneic beta 2-microglobulin are preferentially formed on RMA-S cells cultured at 25 degrees C. These heterologous class I molecules are thermolabile. Increased expression of class I molecules has also been observed on RMA-S cells incubated at 37 degrees C in the presence of class I-restricted peptides. We find that the increased expression of Db molecules induced by influenza virus nucleoprotein residues 365-380 is similarly dependent on culturing RMA-S cells in bovine serum or with purified bovine beta 2-microglobulin.

Animals↗

PubMed1991

Weak base amines can inhibit class I MHC-restricted antigen presentation.

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.

Amines↗

PubMed1991

Reassociation with beta 2-microglobulin is necessary for Db class I major histocompatibility complex binding of an exogenous influenza peptide.

A synthetic peptide corresponding to residues 365-380 of the influenza nucleoprotein (NP365-380) has been previously shown to associate with class I major histocompatibility complex-encoded molecules and to stimulate cytotoxic T lymphocytes [Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. & McMichael, A. J. (1986) Cell 44, 959-968]. We find that intact Db class I heterodimers on the cell surface are unreceptive to binding this antigen. However, NP365-380 readily associates with Db molecules on the plasma membrane in the presence of exogenous beta 2-microglobulin. In addition, there is a second pathway through which this peptide associates with class I molecules that requires energy and de novo protein synthesis. These findings have implications for maintaining the immunological identity of cells and for the use of peptides as vaccines for priming cytolytic T-cell immunity.

Animals↗

PubMed1991

When all is said and done....

Dr. Benacerraf describes his experience in training immunologists for a period of over 30 years. He then analyzes the circumstances in which selected discoveries were made in his laboratory and the events and reasoning that led to these findings. The following topics are discussed: The phagocytic activity of the reticulo-endothelial system. The pathogenesis of immune complex diseases. The impact of activated macrophages on tumor rejection. The evidence that T-cell immunity is specific for internal protein determinants generated by antigen processing. The maturation of the antibody response in terms of antibody affinity. The discovery of Ir gene control of specific immune responses and the role of MHC molecules as presenters of peptide antigens to T cells. The generation of alloreactivity. Lastly, he addresses the administration of science and, particularly, the problems related to the transfer of technology to industrial partners.

Allergy and Immunology↗

PubMed1990

Reassociation with beta 2-microglobulin is necessary for Kb class I major histocompatibility complex binding of exogenous peptides.

T lymphocytes recognize endogenously produced antigenic peptides in association with major histocompatibility complex (MHC)-encoded molecules. Peptides from the extracellular fluid can be displayed in association with class I and class II MHC molecules. Here we report that mature Kb class I MHC molecules bind peptides upon dissociation and reassociation of their light chain. Intact Kb heterodimers, unlike class II MHC molecules, are relatively unreceptive to binding peptides. This property may maintain segregation of class I and class II MHC-restricted peptides and has implications for the use of peptides as vaccines.

Animals↗

PubMed1979

Antigen- and receptor-driven regulatory mechanisms. I. Induction of suppressor T cells with anti-idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

Animals↗

PubMed1979

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

Animals↗