Biomedical subjects

B C Langton

Publications and source records attributed to B C Langton.

16 recordsLinked to original sources

PubMed1996

Growth factor-independent proliferation of rat mammary carcinoma cells by autocrine secretion of neu-differentiation factor/heregulin and transforming growth factor-alpha.

Serially transplantable rat mammary tumor (RMT) cells are not dependent on exogenous epidermal growth factor (EGF) and insulin-like growth factor-I for continuous growth in serum-free medium. Previously, we found that conditioned medium obtained from these cells contained EGF-like mitogenic activity and stimulated tyrosine phosphorylation of a 185-kDa protein in EGF-dependent mammary epithelial cells. This protein is distinct from the EGF receptor and resembles a 185-kDa tyrosine-phosphorylated protein present in RMT cells themselves. The results of the studies reported here indicate that the tyrosine-phosphorylated p185 detected in growth factor-independent RMT cells and in human mammary epithelial cells exposed to RMT-conditioned medium was activated erbB-2 protein. Partial purification of the activating factor present in RMT-conditioned medium yielded a heparin-binding growth factor with biochemical properties similar to those of neu differentiation factor/heregulin (NDF/HRG). RNA-polymerase chain reaction analysis demonstrated that RMT cells expressed mRNA for NDF/HRG, and western-blot analysis confirmed the presence of the 45-kDa secreted form of NDF/HRG in conditioned medium from the growth factor-independent RMT cells. The biological activity of partially purified rat NDF/HRG was examined and found to be the same as that of the pure growth factor. In addition, we found that RMT-conditioned medium, fractionated on an anion-exchange column and by reverse-phase high-pressure liquid chromatography, contained a potent EGF-like growth factor that was distinct from NDF/HRG. This factor competes with 125I-EGF for binding to EGF receptors and has an apparent molecular mass of 6600 Da. This factor copurifies by high-pressure liquid chromatography with pure transforming growth factor-alpha (TGF-alpha), and the cells are positive for TGF-alpha mRNA. Thus, growth factor-independent RMT cells also synthesize and secrete TGF-alpha. These results indicate that growth factor-independent cells secrete two growth factors with overlapping biological activities and suggest that autocrine loops mediated by these factors are important in the growth factor-independent proliferation of the RMT cells.

Animals↗

PubMed1995

Construction of a chimeric antibody with therapeutic potential for cancers which overexpress c-erbB-2.

We describe the chimerization of a monoclonal antibody directed against the c-erbB-2 protein using a novel PCR method for cloning immunoglobulin variable region genes. We also describe the characterization of the chimera and show its potential use for treating cancers which overexpress the c-erbB-2 protein. The genomic DNA fragments of heavy and light chain variable genes were cloned by PCR using uniquely designed primers which allowed for isolation of genes containing functional promoters, signal and coding sequences. The chimeric genes were then constructed by linking variable regions of murine genes to human constant gamma 1 and kappa genes. Expression of the chimeric immunoglobulin genes resulted in production of properly assembled chimeric antibody with improved biological properties.

Animals↗

PubMed1992

Expression of human transforming growth factor alpha by Chinese hamster ovarian tumors in nude mice causes hypercalcemia and increased osteoclastic bone resorption.

Transforming growth factor alpha (TGF-alpha) is a polypeptide regulator of cell growth produced by many malignant tumors. It stimulates osteoclastic resorption in bone organ culture and osteoclast-like cell formation in marrow culture. To determine whether tumor production of TGF-alpha can cause hypercalcemia in vivo, we used Chinese hamster ovarian (CHO) cells transfected with the human TGF-alpha gene (TCHO), which stably express and secrete TGF-alpha. We used nontransfected CHO cells as controls (CCHO). TCHO and CCHO were inoculated intramuscularly into one hindlimb of nude mice and grew as local solid tumors. After 4 weeks of TCHO tumor growth, plasma ionized calcium (Ca2+) increased to reach 1.48 +/- 0.03 mM (mean +/- SEM), whereas mice bearing similarly sized CCHO tumors and non-tumor-bearing mice (NTB) remained normocalcemic (normal range for Ca2+, 1.15-1.30 mM). Plasma TGF-alpha was undetectable by an ELIFA assay in all NTB mice, was markedly increased in all TCHO mice (5.75 +/- 0.78 ng/ml), and was slightly increased in CCHO mice (0.50 +/- 0.22 ng/ml). Quantitative bone histomorphometry showed a prominent increase in osteoclastic bone resorption in TCHO mice. These data suggest that TGF-alpha is a mediator of hypercalcemia and increased osteoclastic bone resorption in tumors that produce it in sufficient quantity.

Animals↗

PubMed1992

Elevated soluble c-erbB-2 antigen levels in the serum and effusions of a proportion of breast cancer patients.

PURPOSE: An enzyme-linked immunosorbent assay (ELISA) for the extracellular domain of the c-erbB-2 oncogene product was developed and evaluated to determine if soluble c-erbB-2 could be detected in the serum and effusions of cancer patients. PATIENTS AND METHODS: Sera from 208 previously untreated or progressing cancer patients and 69 healthy controls were assayed in a double-antibody sandwich ELISA that used two monoclonal antibodies to the native extracellular domain of the c-erbB-2 receptor. Fisher's exact test was used to analyze the statistical significance of the frequency of elevated serum c-erbB-2 levels. Immunoprecipitation and Western blotting were used to characterize further the c-erbB-2 immunoreactivity in the serum of four breast cancer patients. RESULTS: Sera from 12 of 53 patients (23%) with metastatic or locally advanced breast cancer, zero of 69 controls, one of 31 patients with ovarian cancer (3%), and two of 124 other cancer patients (2%) had soluble c-erbB-2 values greater than or equal to 5 U/mL. The number of breast cancer patients with elevated serum c-erbB-2 levels was significantly greater than that of the control group (P less than .0001), the ovarian cancer group (P less than .03), and the other cancers group (P less than .0001). Also, two of five effusions (40%) from breast cancer patients had an elevated soluble c-erbB-2 antigen level, compared with zero of 17 effusions from patients with benign diseases. Western blotting of four sera from breast cancer patients with elevated serum c-erbB-2 antigen levels produced bands of approximately 105 kD that seemed to correlate in intensity with increasing ELISA serum levels. CONCLUSION: Serum c-erbB-2 levels are elevated in approximately one fourth of patients with locally advanced or metastatic breast cancer.

Adult↗

PubMed1991

A monoclonal antibody against the c-erbB-2 protein enhances the cytotoxicity of cis-diamminedichloroplatinum against human breast and ovarian tumor cell lines.

A monoclonal antibody (TAb 250) specific to an extracellular epitope of the c-erbB-2 protein (gp185) inhibited the in vitro proliferation of human breast tumor cell lines that overexpress c-erbB-2 in a dose-dependent manner. Treatment of cells with combinations of cis-diammedichloroplatinum (CDDP) and TAb 250 resulted in a significantly enhanced cytotoxic effect. This synergistic cytotoxicity was apparent over a wide range of antibody concentrations (200 pg/ml-100 micrograms/ml) including concentrations that showed no inhibitory effect alone. TAb 250 did not increase the cytotoxic effect of CDDP in a cell line exhibiting no detectable level of gp185. Athymic mice bearing s.c. xenografts of human tumor cells expressing high levels of gp185 showed a greatly enhanced inhibition of tumor growth when treated with TAb 250 and CDDP compared to treatment with the antibody or CDDP alone. This effect was specific inasmuch as TAb 250 did not enhance the growth-inhibitory effect of CDDP on tumor xenografts which were not expressing gp185.

Animals↗

PubMed1991

An antigen immunologically related to the external domain of gp185 is shed from nude mouse tumors overexpressing the c-erbB-2 (HER-2/neu) oncogene.

An antigen, immunologically related to the external domain of the c-erbB-2 (HER-2/neu) protein, was found shed into the serum of nude mice bearing tumors that overexpress the c-erbB-2 protein (gp185). Utilizing paired combinations from a panel of monoclonal antibodies (TAbs 250-265), with specificity for extracellular epitopes of gp185, an immunoradiometric assay was developed to quantitate this shed antigen. The immunoradiometric assay detected membrane-bound and soluble gp185 as well as a soluble derivative corresponding in sequence to the extracellular domain of gp185 (designated gp75). This recombinantly expressed gp75 was immunoaffinity purified and used to generate a standard curve from which serum samples were quantitated. Increases in antigen levels measured in the sera of tumor-bearing nude mice correlated with both overexpression of the c-erbB-2 protein and increased tumor volume. Positive sera were obtained from mice given implants of NIH3T3 cells transfected with c-erbB-2 complementary DNA (NIH3T3t), or ovarian (SK-OV-3) or breast (MDA-MB-361) tumor cell lines overexpressing the c-erbB-2 protein. In mice bearing NIH3T3t tumors, increases in tumor volume from 80 to 9000 mm3 resulted in levels of shed antigen from 8 to greater than 1000 ng/ml gp75 equivalents. Sera from mice with c-erbB-2-negative tumors or tumors overexpressing the epidermal growth factor receptor were negative in the assay. This assay, and the quantitation of shed antigen levels, may have diagnostic or monitoring utility in cancers, such as breast and ovarian, in which the c-erbB-2 protein is overexpressed.

Animals↗

PubMed1991

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.

Amino Acid Sequence↗

PubMed1990

Increased erbB-2 gene copies and expression in multiple stages of breast cancer.

In order to examine the role of the erbB-2 oncogene in human breast cancer, gene amplification and expression were examined in multiple stages of tumor progression. Gene amplification ranging from 2-fold to 32-fold was found in 30 (29%) of 130 cases analyzed. Expression of the receptor-like gene product was determined by a combination of Western immunoblotting and immunohistochemistry. In each case of gene amplification, there was high level overexpression (+ + +) of the protein product. In an additional 29 of 111 cases in which expression was studied (26%), there was moderate level overexpression (+ +) of erbB-2 in the absence of gene amplification. Amplification and overexpression of the erbB-2 gene were found in early clinical stages of breast cancer as well as in more advanced cases. In 23 patients, gene number and level of gene expression were equivalent in the primary tumor site compared with single or multiple metastatic sites in regional lymph nodes. Using a combination of immunohistochemistry and in situ cytohybridization, high (+ + +) and moderate (+ +) level overexpression were homogeneously present in all malignant epithelial cells within histological sections of both primary and metastatic tumor. The intraductal component of carcinoma was identified in sections from 16 invasive primary tumors. erbB-2 gene expression in the intraductal lesions was equivalent to or exceeded expression in the infiltrating components of these tumors. Because erbB-2 alterations are (a) present in all clinical stages, (b) maintained during metastatic spread, (c) homogeneously present throughout tumor sections, and (d) present in the in situ as well as infiltrating component, we conclude that these alterations are selected for early and may be important in the initiation of certain mammary cancer.

Breast Neoplasms↗

PubMed1990

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation.

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.

Blotting, Northern↗

PubMed1989

Identification of immunodominant regions of transforming growth factor alpha. Implications of structure and function.

Human transforming growth factor alpha (TGF alpha) is a 50-residue mitogenic peptide with a compact structure restrained by three disulfide bonds. Sequential and overlapping synthetic peptides were made to identify epitopes of TGF alpha using a panel of murine monoclonal antibodies and rabbit polyclonal antibodies. Antibodies were raised against human TGF alpha from different preparations obtained from either chemical synthesis or recombinant DNA techniques. Two related methodologies were used in these experiments. In the first method, probes were synthesized as peptides immobilized on polyethylene pins by the method of Geysen et al. (Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002). Three sets of sequentially overlapping tetrapeptides, hexapeptides, and octapeptides covering the entire length of the human TGF alpha sequence were synthesized. In the second method, a set of overlapping 8-residue synthetic peptides, freely soluble in solution, were used as probes. By both methods, the nonneutralizing monoclonal antibodies, i.e. those that did not inhibit TGF alpha in mitogenic assays, recognized two immunodominant regions represented by the NH2-terminal segment (residues 1-9) and the most prominent beta-sheet of the molecule (residues 22-31). The NH2 terminus and the beta-sheet-(22-31) are in the same face of the molecule as determined by the solution structure. These two immunodominant regions were also recognized by the polyclonal antibodies as well as regions in the COOH terminus as minor epitopes. However, none of the neutralizing monoclonal antibodies recognized any synthetic peptides. Thus, our results suggest that the receptor-binding surface of TGF alpha does not involve the face represented by the NH2-terminal fragment and the major beta-sheet of residues 22-31, but rather, that the opposite face represented by two loops formed by residues 12-20 and 34-43 may be involved in TGF alpha binding to its receptor.

Amino Acid Sequence↗

PubMed1989

Transformation of an established mouse mammary epithelial cell line following transfection with a human transforming growth factor alpha cDNA.

To determine whether the enhanced expression of transforming growth factor alpha (TGF alpha) is sufficient to induce the neoplastic transformation of an immortalized population of mammary epithelial cells, we cotransfected NOG-8 cells, a cloned mouse mammary epithelial cell line, with a simian virus 40-human TGF alpha cDNA expression vector plasmid and a pSV2neo plasmid. After cotransfection, nine G418-resistant NOG-8 colonies were cloned and expanded. All clones were subsequently analyzed for TGF alpha mRNA expression by northern blot analysis, TGF alpha secretion, anchorage-dependent growth in serum-free medium, anchorage-independent growth in soft agar, and tumorigenicity in nude mice. Three TGF alpha-transfected NOG-8 clones expressed high levels of a specific TGF alpha mRNA, secreted elevated levels of TGF alpha into the culture medium (177-595 ng/10(8) cells/48 h), exhibited an enhanced growth rate, grew aggressively as colonies in soft agar, and formed undifferentiated, invasive carcinomas in nude mice. A neutralizing mouse monoclonal antibody generated against the low molecular weight human TGF alpha peptide was able to inhibit colony formation in soft agar by TGF alpha-transfected NOG-8 clones that produced high levels by TGF alpha. This inhibition suggested that TGF alpha acted through an external autocrine loop. NOG-8 cells and NOG-8 cells transfected with a pSV2neo plasmid alone secreted very low levels of TGF alpha, failed to grow as colonies in soft agar and did not form tumors in nude mice. These results demonstrate that overexpression of a human TGF alpha cDNA in immortalized, nontransformed mouse mammary epithelial cells can induce a transformed phenotype in vitro and can facilitate tumor formation in vivo.

Animals↗

PubMed1988

Structural features of an antigen required for cellular interactions and for T cell activation in a MHC-restricted response.

The protein Ag, tobacco mosaic virus protein, (TMVP) and its tryptic peptide number 8 (residues 93-112 of the protein) exhibit cross-reactivity on the T cell level in some strains of mice (e.g., C3H.SW, C57BL/10); these strains are termed cross-reactive (CR). In other strains such as A/J or B10.BR, no cross-reactivity is exhibited; these strains are termed non-cross-reactive (NCR). Genetic experiments indicated that the cross-reactivity is dominant and that it is mapped to the I-A or I-E region of the MHC, with cross-reactivity exhibited by the I-Ab haplotype but not by I-Ak or I-Ek. Cell reconstitution experiments have indicated that the non-cross-reactivity is associated with the inability of the NCR APC to present Ag. Analysis of the area(s) on peptide 8 which serve(s) as epitope revealed that both strains recognize an overlapping area consisting of 11 amino acid residues in the middle of peptide 8 (residues 97-107), which by itself is nonstimulatory to TMVP- or peptide 8-immune T cells of the CR or the NCR strains. However, the addition of a few amino acid residues of the sequence of peptide 8 to this area converts it to a complete stimulatory epitope. Additivity experiments revealed that the CR strain contains two major T cell populations each recognizing this middle region of peptide 8 when elongated by a few amino acids N-terminally and C-terminally, respectively. In contrast, the NCR strain contains one major T cell population recognizing elongation only N-terminally. Because TMVP (but not peptide 8) requires processing before presentation to T cells, it is postulated that, during processing of TMVP, there occur alterations in the area of the proximal three or four N-terminal amino acids of the region consisting of peptide 8, destroying the only region containing the T cell epitope recognized by the NCR strain, hence TMVP and peptide 8 do not exhibit cross-reactivity in this strain. The same alterations of TMVP still leave intact an epitope consisting of amino acid residues C-terminal to the altered area which is recognized by the CR strain, hence the cross-reactivity exhibited by this strain. The results suggest that the difference in cross-reactivity on the T cell level between TMVP and peptide 8 exhibited by the strains may be due to differences in the orientation of presentation and the subsequent cell recognition of an epitope contained within peptide 8.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1987

Structural considerations of T-cell cognizance of antigen.

Work summarized herein describes two congenic strains of mice, C57BL/10 and B10.BR, which differ at the I-A region of the MHC where C57BL/10 and B10.BR are I-Ab and I-Ak respectively. Using various antigens to stimulate, in vitro, primed T lymphocytes, studies on the responsiveness of these two strains to immunization with TMVP and with its tryptic peptide number 8 representing residues 93-112 of the protein are described. These studies revealed that in the C57BL/10 strain TMVP and peptide 8 exhibit cross reactivity on the T-cell level, whereas in the B10.BR strain these two antigens do not exhibit such cross reactivity. Further studies revealed that the cross reactivity is mapped to the I-A region of the MHC with cross reactivity between TMVP and peptide 8 exhibited in mice with I-Ab (referred to as cross reactive-CR) but not with I-Ak (referred to as non cross reactive-NCR). Cell depletion and reconstitution experiments revealed that cross reactivity (or the lack thereof) could be attributed to antigen presenting cells (APC). A profound difference between the strains was also expressed on the level at which their immune T-cells reacted in vitro to stimulation by various derivatives of peptide 8. The studies suggest that the strains differ in their responsiveness to the various orientations in which the T-cell epitope(s) are presented. Assuming that TMVP, but not peptide 8, is altered during processing by APC and assuming that antigen processing by APC of CR and NCR is similar, the difference which the strains exhibit in recognizing different orientations of the epitope may account for the difference in the cross reactivity between TMVP and peptide 8 exhibited by these strains. Further investigations revealed that the strains also differ in their response to pre-immunization with TMVP prior to immunization with peptide 8. Whereas preimmunization of the C57BL/10 (CR) mice with TMVP did not affect subsequent response to immunization with peptide 8, preimmunization of B10.BR (NCR) mice with TMVP suppressed the response to subsequent immunization with peptide 8. It is suggested that the presentation of the T-cell epitope(s) in the correct orientation by the appropriate Ia is crucial for the activation of T helper cells but that such presentation does not play a decisive role in the activation of T suppressor cells.

Animals↗

PubMed1986

Antigenic requirements for T-cell activation: reconstitution of a functional antigen from an inactive peptide portion of an antigen conjugated to protein carriers.

The structural features of an antigenic peptide required for T-cell activation were examined by a novel approach: an active antigen was constructed from an inactive peptide portion of the original antigen by conjugating it to various proteins. An eicosapeptide, peptide 8, representing residues 103-112 of the tobacco mosaic virus protein (TMVP), was utilized as the model antigen for these studies. While peptide 8 was able to stimulate, in vitro, T-cells from peptide 8 primed mice, synthetic peptides representing various portions of peptide 8 were unable to activate these cells. Although the amino-terminal undecapeptide of peptide 8 (residues 93-103 of TMVP) was unable to activate T-cells from peptide 8 primed mice, conjugates which consisted of this undecapeptide coupled to certain proteins were capable of inducing antigen-specific proliferation of these T-cells. These results identify two structural antigenic features essential for T-cell activation: a T-cell-recognizable epitope within the amino-terminal undecapeptide of peptide 8 and a second region provided by the carboxy-terminal half of peptide 8 or by protein carriers. Potential roles for this second region include providing a site for antigen interaction with Ia molecules on the antigen-presenting cell or, alternatively, providing amino acids important in stabilizing the binding of the T-cell antigen receptor. The results suggest that the recognition of this second region exhibits only a limited specificity.

Animals↗

PubMed1986

Cell activation and immunogenicity.

There is presently great interest in the production of synthetic vaccines which utilize as immunogens peptides representing portions of protein antigens, either free or conjugated to protein carriers. The use of such immunogens raises questions regarding the cells which are activated an the characteristics of the resulting immune response. Using the tobacco mosaic virus protein (TMVP) as a model antigen, immune induction by the protein and by synthetic vaccines related to this protein was investigated. Specifically we have compared immune induction by the parent protein, an unconjugated eicosapeptide representing residues 93-112 of the protein, and an immunogen consisting of the C-terminal decapeptide of the above eicosapeptide conjugated to the protein carrier KLH. The comparison led to the following conclusions: Immunization with the free eicosapeptide but not with its C-terminal decapeptide leads to the activation of T and B lymphocytes. Immunization with the free eicosapeptide or with its C-terminal decapeptide-KLH conjugate induces antibodies capable of reacting with the parent protein. The isotype composition of the antibodies induced by these immunogens is similar to that induced by immunization with the whole protein. The fine specificity of the antibodies induced by all three immunogens is similar. However, the antibody populations induced by the synthetic immunogens may be devoid of one or more clonotypes depending upon constraints imposed by cellular interaction. Antigen specific T helper cells do not seem to influence the fine specificity of antibodies induced to a given epitope. Comparison of the induction of memory responses by the three immunogens led to the conclusion that immunization with the peptide hapten conjugated to the heterologous carrier KLH does not lead to an anamnestic antibody response upon encounter with the native protein. Immunization with an immunogenic peptide representing a portion of the protein recognized by T and B lymphocytes leads to an anamnestic antibody response upon encounter with the native protein.

Animals↗

PubMed1985

Immune induction by a protein antigen and by a peptide segment of the protein.

The immune induction by a protein (the tobacco mosaic virus protein-TMVP) was compared to the immune induction by the free, non-conjugated eicosa tryptic peptide fragment of the protein (tryptic peptide 8 representing residues 93-112 of the protein). The results demonstrated that like TMVP, peptide 8 was immunogenic in A/J mice. TMVP and peptide 8 do not cross react on the T cell level. However, immunization with TMVP or with peptide 8 induces antibodies which react with both TMVP and peptide 8. Characterization of the antibodies produced by both immunogens revealed that: their isotope composition is similar with IgG1 and IgG2 being the predominant isotypes; this composition indicates that both immunogens are T cell dependent antigens, the antibodies induced by TMVP and by peptide 8 are directed against the C-terminal decapeptide portion of peptide 8 (residues 103-112 of the protein), the fine specificity of these antibodies is the same. These results, and results of adoptive transfer experiments, indicate that antigen specific T cells had no effect on the expression of the fine antibody specificity. The results demonstrate the feasibility of immunizing with a portion of a protein for the purpose of inducing antibodies with the same isotype composition and specificity towards a protein epitope as those induced by immunization with the whole protein.

Amino Acid Sequence↗