Biomedical subjects

M J Solvay

Publications and source records attributed to M J Solvay.

10 recordsLinked to original sources

PubMed1988

Functional and biochemical parameters of peptide antigen presentation.

To understand the mechanism by which peptide antigens are processed and presented to T cells, we examined the T-cell response to the 13-amino-acid peptide alpha-melanocyte-stimulating hormone (alpha-MSH). To determine the fine specificity of T-cell recognition, T cells specific for alpha-MSH, and genetically restricted by I-Ab/d, were challenged with different alpha-MSH analogs and homologs. It was found that intact alpha-MSH, including the blocked amino and carboxy termini of the native molecule, was required for T-cell responsiveness. Antigen-presenting cells (APC) could be briefly pulsed with alpha-MSH and then present the alpha-MSH antigenic determinant to T cells, indicating that the relevant antigen was retained by the APC. APC stimulatory capacity was dramatically reduced by aldehyde treatment of the APC, or by pulsing the APC with alpha-MSH at low temperature. Efficient alpha-MSH pulsing was also impaired by treatment of the APC with the carboxylic ionophore, monensin, but not by the lysosomotropic agents chloroquine and methylamine. In addition, isolated APC plasma membranes added to the T cells in the presence of soluble alpha-MSH were not stimulatory. However, plasma membranes isolated from APC that had been previously pulsed with alpha-MSH retained stimulatory activity for T-cell responses. The only detectable alpha-MSH contained in these pulsed APC membranes was in an acid-stable complex of higher molecular weight than native peptide. The amount of alpha-MSH detected in the cellular membrane fraction isolated by density gradient sedimentation was also reduced by treatments that reduced the APC stimulatory capacity, such as pulsing at low temperature or in the presence of monensin. Taken together, these results suggest that processing of alpha-MSH is unlike that heretofore described for other peptide antigens and seems to involve APC handling to form the stimulatory moiety presented on the APC surface.

Animals↗

PubMed1987

Heterogeneity in cellular antigen retention structures.

The mechanism of presentation of foreign antigens to helper T lymphocytes and the nature of the structures involved in this process are not totally understood. It is well documented that this event is carried out by antigen-presenting cells (APC) (e.g., macrophages, dendritic cells, and B lymphocytes) that internalize the antigen, process it, reexpress it on their membrane surface, and present it to the T cell in the context of major histocompatibility complex class II (Ia) molecules. Recent evidence supports the hypothesis that peptide antigens associate directly with Ia molecules on the APC surface membrane. However, the characteristics of other APC membrane structures potentially involved in antigen presentation are not entirely clear. Previous studies in our laboratories identified a guinea pig macrophage membrane-bound, non-Ia-containing antigenic complex (peak A) formed upon incubation of APC with the octapeptide antigen angiotensin (AII). This complex was capable of stimulating AII-immune guinea pig T cells and thus appeared to contain the immunologically relevant form of the antigen. For this reason it was important to establish whether such complex formation with peptides occurs with other cell types and with other peptide antigens. In the present study we found that other types of cells are also capable of forming such a membrane complex with antigen (peak A) and that this event is not unique to AII. Two other peptides, alpha-melanocyte-stimulating hormone and human fibrinopeptide B, both of which are antigenic in mice, were found to form peak A with a number of murine cell lines. As in our earlier studies with guinea pig macrophages, there was no evidence from these experiments for a role for major histocompatibility complex Ia antigens in the peptide binding observed. Differences in both the amount of peak A formation and the pattern of peptide antigen degradation were found from cell line to cell line for a given peptide, and from peptide to peptide for a given cell line, suggesting cellular heterogeneity in peptide processing and retention. In addition, cross-inhibition studies indicated that there was peptide specificity in the formation of peak A perhaps suggestive of molecular heterogeneity in the structure of peak A. These results indicate that there may be several types of cell surface molecules that specifically bind and retain peptide antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1986

T lymphocyte recognition of insolubilized peptide antigen.

To study the role of antigen-presenting cells (APC) in T lymphocyte responses, the stimulation requirements of a murine T cell hybridoma specific for the peptide antigen human fibrinopeptide B (hFPB)/I-Ak was examined. The fine specificity of T cell recognition of this peptide was determined by using several hFPB homologs and analogs, which indicated that the intact 14-amino acid peptide must remain intact to preserve the antigenic determinant, and that the carboxyl terminal Arg14 was important for T cell responses. Of particular interest was the finding that APC-associated hFPB failed to stimulate the T cells, and that activation was only observed with soluble peptide or by brief hFPB treatment of the T cells and APC mixed together. In addition, hFPB covalently bound to agarose beads was able to cause T cell activation, provided that I-Ak+ APC were also present in the culture. A number of control experiments were performed that showed that hFPB was not released from the bead and that the antigenic peptide involved in T cell responses remained bound to the beads. These results indicate that the form of the hFPB peptide antigen recognized by this T cell can be provided separately from APC.

Animals↗

PubMed1986

Direct stimulation of T lymphocytes by antigen-conjugated beads.

To examine T lymphocyte recognition of foreign antigen, specific responses to the photoreactive antigen N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) were determined by using an HSAB/I-Ad-reactive murine T cell hybridoma. It was found that covalent coupling of HSAB to aminoethyl polyacrylamide beads at particular densities directly activated the T cells for IL 2 production, and beads conjugated at higher or lower doses of HSAB were nonstimulatory. This stimulation was specific for the phenyl ring composition of HSAB and for HSAB-reactive T cells. In addition, T cell activation by HSAB-coupled beads was specifically inhibited by soluble monomeric HSAB-glycine. These results indicate that HSAB-specific T cells may be directly stimulated by insolubilized HSAB in the absence of Ia antigens, suggesting direct T cell binding of foreign antigen.

Animals↗

PubMed1985

Cellular requirements for antigen processing by antigen-presenting cells: evidence for different pathways in forming the same antigenic determinants.

In this report we examined the antigen-presenting cell (APC) requirements for activation of T-cell hybridomas specific for the protein antigen PPD (purified protein derivative of tuberculin). During the course of these studies we observed that glutaraldehyde fixation of Ia-positive A20.2JAD (A20) and P388D1 stimulator cells had different effects on T-cell activation. A20 cells fixed with glutaraldehyde stimulated the T cells in the presence of PPD as efficiently as nonfixed A20 cells. By contrast, glutaraldehyde treatment of Ia-positive P388D1 cells dramatically inhibited their ability to process and/or present PPD to T cells. This was not due to nonspecific effects on the P388D1 cells since cells prepulsed with PPD prior to glutaraldehyde treatment stimulated T cells as efficiently as non-glutaraldehyde-treated P388D1 cells. In addition, there was no apparent difference in "fixing" of the two cell types as determined by the uptake of radiolabeled thymidine. These observations suggested that P388D1, but not A20, cells required PPD internalization to form the relevant antigenic determinants. This was substantiated by showing that treatment of P388D1 cells with chloroquine prior to PPD pulsing eliminated their stimulatory capacity, but had no effect on P388D1 cells previously pulsed with PPD. Chloroquine treatment had no effect on stimulation by A20 cells. Since PPD internalization appeared not to be required for presentation by A20 cells, we next determined if isolated A20 plasma membranes would substitute for the intact cell. We observed that the isolated plasma membranes from PPD-pulsed A20 cells stimulated the T hybridoma cells, and that this stimulation was antigen-specific and was inhibited by anti-Ia monoclonal antibodies. Taken together, the results presented here suggest that for the PPD-specific T-cell responses examined here, different APC utilize distinct pathways to present the same antigenic determinant for T-cell recognition.

Animals↗

PubMed1985

Modulation of Ia and photoreactive antigen on antigen-presenting cells: fun with a photoprobe.

To identify the antigen-specific recognition complex containing elements from T cells and antigen-presenting cells (APC), a photoactivatable antigen system was developed which could potentially crosslink the complex during the specific cellular responses. In this paper we describe the development of this system using murine T-cell hybridomas responding to stimulator cells chemically conjugated with N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and genetically restricted by I-Ad. In initial experiments it was found that several I-Ad-positive B-cell lines were nonstimulatory when coupled with HSAB, but that I-Ad-positive P388D1 macrophage-like cells were efficient stimulators of HSAB-specific T-cell responses. These results suggested that the relevant HSAB coupled surface structure was not likely I-Ad. To substantiate this point, Ia-positive or Ia-negative P388D1 cells were initially coupled with HSAB and the expression of Ia was modulated by the addition and withdrawal of Con A-stimulated spleen cell supernatant fluid through several days of culture. Under these conditions, efficient stimulation was only observed when Ia was expressed, although the HSAB antigen was continuously present. In other experiments it was found that exposure of HSAB-coupled APC to light selectively eliminated their stimulatory capacity for HSAB-specific T hybridomas, suggesting that the light-induced crosslinking by HSAB directly eliminates the antigenic determinant. This antigen system allows a unique opportunity to manipulate the antigen during specific cellular interactions, and to introduce covalent crosslinking of the specific antigen recognition complex that may allow its isolation and characterization.

Animals↗

PubMed1985

Importance of the COOH terminal of angiotensin in antigenicity and in the formation of an antigen-containing complex with cellular membrane structures.

To more carefully determine how a peptide antigen interacts with the antigen-presenting cell (APC), we have begun an analysis of the fate of APC-associated peptide antigens. These studies have shown that a stable cell-bound form of APC-associated peptide exists, which is a complex of the peptide with surface membrane structures (peak A). In the experiments described here, we have begun to examine the chemical mechanism of this peak A complex formation. By modifying either the carboxyl terminal or amino terminal group of the octapeptide antigen angiotensin II we have established that the terminal carboxyl group, but not the terminal amino group, was critical for forming the peak A complex with APC membrane structures. In addition, blocking the carboxyl but not the amino terminal dramatically reduced the antigenicity of the peptide for AII-immune T cell in vitro proliferation. These results show that the carboxyl terminal of AII is essential for both peak A formation and antigenicity, and suggest that peak A is critical for antigen presentation to T cells.

Acetylation↗

PubMed1984

Macrophage processing of peptide antigens: identification of an antigenic complex.

In this report, we present data from our examination of the fate of the octapeptide antigen angiotensin II (AII) after processing by guinea pig macrophages. Macrophages were cultured with [125I]-AII or [3H]-AII for various times, and the AII products in the culture supernatant fluid and solubilized cell lysate (macrophage-associated AII) were analyzed by chromatography on Sephadex G-25. With [3H]-AII, both the cell-associated and cellfree AII was catabolized to three distinct peptide fragments: Peak B, containing seven to eight amino acids; Peak B1, with five to six amino acids; and Peak C, containing four to five amino acids. Surprisingly, in the cells another major form of AII was obtained, Peak A, which showed an m.w. of 50,000 to 70,000. Formation of Peak A was blocked by inclusion of non-radiolabeled AII into the incubation mixture, and could be formed by repulsing fresh macrophages with isolated Peaks B and B1, and to only a minor extent with Peak C. Peak A was formed within an hour of culture of macrophages with [3H]-AII, and remained stably cell-associated through a day of culture. In a pulse-chase type of experiment, it was shown that macrophages rapidly exocytose cell-associated Peaks B and C, and Peak B1 less rapidly, whereas Peak A is retained and is the predominant cell-associated form of AII after a day in culture. Incubation of [3H]-AII with isolated macrophage plasma membranes also resulted in Peak A formation, showing that the complex can be formed by a membrane event not requiring cellular uptake of peptide. The complex of AII in Peak A was acid-stable, and approximately 20% of Peak A was recovered after boiling in SDS. Because Peak A is the primary form of AII retained by macrophages, it was important to determine if this complex was antigenic for T cells. AII-immune T cells cultured with Peak A showed significant proliferation that was specific for AII. Thus, Peak A appears to represent a stable complex of AII with macrophage membrane structures that provide an immunologically relevant form of the antigen. These results are discussed with respect to a pathway for macrophage processing of peptide antigens to form a stable membrane-bound complex that is important for T cell responses.

Amino Acid Sequence↗

PubMed1979

Recognition of viral antigens in 6/94 virus-induced T-cell-mediated cytotoxicity.

Distinct events in the virus-stimulated T-cell-mediated cytotoxicity (V-CMC) have been investigated: 1.) The induction of V-CMC is possible by immunizing mice with infectious as well as UV-inactivated virus (parainfluenza type 1 strain 6/94), or with virus-infected cells either compatible or imcompatible with the recipient. 2). Recognition of viral antigens by the effector cells occurs independently of the H2 environment: Fractionation of effector cells on columns loaded with virus-infected cells eliminates virus-specific cytotoxic cells. Effector cells and cells on the column need not share H-2 antigens. The findings are discussed with regard to the H2 restriction of the virus induced T-cells mediated cytotoxicity.

Animals↗

PubMed1978

Cell-mediated cytotoxicity: comparison of primary and secondary immune reactions after parainfluenza type 1 virus inoculation.

H-2 antigen compatibility of effector spleen cells and target cells was necessary for cell-mediated cytotoxicity (CMC) after both 1 and 2 intraperitoneal inoculation of 6/94 virus into mice. T cells were responsible for both primary and secondary CMS reactions. Neither reaction was influenced by the presence of virus-specific antibody added to the test system. Secondary CMC peaked earlier than the primary response and declined more rapidly. In vitro stimulation of primed spleen cells could also be used to detect the secondary CMC response.

Animals↗