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Biomedical subjects

S Ferrone

Publications and source records attributed to S Ferrone.

At least 73 records · Page 4Linked to original sources
PubMed1997

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens.

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.

Animals↗
PubMed1997

Control of B cell lymphoma recognition via natural killer inhibitory receptors implies a role for human Vgamma9/Vdelta2 T cells in tumor immunity.

The Vgamma9/Vdelta2 T cell receptor (TCR) is expressed by most human gammadelta T cells. We show here that cytotoxic T lymphocytes of the Vgamma9/Vdelta2 subset, but not of the Vdelta1 subset of human gammadelta T cells, express natural killer inhibitory receptors (KIR) with specificity for different HLA class I alleles that down-regulate TCR-mediated signaling in response to HLA class I-expressing B cell lymphomas. Vgamma9/Vdelta2 T cell clones with a T helper cell phenotype lack KIR and produce lymphokines in response to most human B cell lymphomas, just as they do upon recognition of the HLA class I-deficient human Burkitt's lymphoma Daudi. Thus, human Vgamma9/Vdelta2 T cells have an innate specificity for nonpolymorphic cell surface structures expressed by many lymphoma cells and their cytotoxic activity is controlled by KIR. These results imply a general role of human Vgamma9/Vdelta2 T cells in the defense against hematopoietic tumors that is distinct from NK cells.

Cells, Cultured↗
PubMed1997

Titration calorimetry study of an anti-idiotypic antibody cascade in a human melanoma-associated antigen system.

The thermodynamic parameters of interactions between six variants of the anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-idiotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-specific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five HMW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 386 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthalpies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibited significantly higher binding enthalpies for the interaction with the anti-idiotypic than the idiotypic mAbs. Replacement of either the heavy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. The values of the binding enthalpy showed only small variations when binding of the idiotypic mAb CGP 76873 to four variants of the anti-idiotypic mAb CGP 60686 with different immunoglobulin constant regions, but identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per binding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interactions. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-driven processes with only minor entropic contributions. High sensitivity titration calorimetry was used to monitor protein-protein interactions within an anti-idiotypic antibody cascade. It was found that the direct measurement of the interaction enthalpy allowed a quantitative characterization of the binding processes studied.

Animals↗
PubMed1997

TAP off--tumors on.

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ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Purification by affinity chromatography with anti-idiotypic monoclonal antibodies of immunoreactive monoclonal antibodies following labeling with 188Re.

Because of its high energy beta emissions and imageable gamma emissions, 188Re represents an attractive isotope to radiolabel monoclonal antibodies (mAb) recognizing human tumor-associated antigens for radioimaging and radioimmunotherapy in patients with malignant diseases. The application of 188Re is, however, hindered by the denaturation of a sizable proportion of antibody molecules during the labeling process. To overcome this problem, we have combined radiolabeling of mAb with 188Re with purification of immunoreactive 188Re-labeled mAb by affinity chromatography over columns of anti-idiotypic (anti-id) mAb. Utilizing the anti-high-molecular-weight melanoma-associated antigen (HMW-MAA) mAb 763.74 as a model system, we found that the immunoreactivity of mAb 763.74 labeled with 188Re to a specific activity of 1 mCi/mg increased from about 50% to at least 80% following passage over columns of immobilized anti-id mAb. Moreover, between 90-100% of immunoreactive mAb contained in radiolabeled preparations could be recovered from anti-id mAb columns. These results indicate that the procedure we have described may facilitate the application of 188Re for immunoscintigraphy and immunotherapy of malignant diseases.

Antibodies, Anti-Idiotypic↗
PubMed1997

Induction of nuclear factor kappa B/Rel nuclear activity in human peripheral blood T lymphocytes by anti-HLA class I monoclonal antibodies.

Monoclonal antibodies against either monomorphic or polymorphic determinants of class I antigen induced in PBMC and highly purified T lymphocytes the nuclear activity of NF-kappa B/Rel complexes. These included both p50/p50 and p50/p65 dimers, recognized by specific antibodies in EMSA. The induced complexes were detectable in extracts of cells incubated with anti-class I monoclonal antibody (mAb) for 1.5 h; the induction was maximal at 5 h, persistent at 16 h and no longer observed at 40 h. The mAb failed to induce NF-kappa B/Rel nuclear activity in cells incubated in the presence of 3,4-dichloroisocoumarin, an inhibitor of I kappa B-alpha degradation. Together, these results suggest that class I triggering can induce the activity of NF-kappa B/Rel nuclear activity in peripheral blood T lymphocytes, thereby modulating the expression of genes regulated by these transcription factors.

Animals↗
PubMed1997

Characterization of rabbit antisera elicited with human LMP2- and LMP7-specific peptides and recombinant proteins.

Anti-human LMP2 and anti-human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2- and LMP7-specific peptides corresponding to C-terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP-specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross-reactive with the other one. This antibody subpopulation was not detectable in the affinity-purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti-LMP2 nor anti-LMP7 sera displayed cross-reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity-purified anti-LMP2 and anti-LMP7 antibodies stained cells in both frozen and formalin-fixed tissue sections of normal skin. These results indicate that the anti-LMP2 and anti-LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.

Amino Acid Sequence↗
PubMed1997

Beta2-microglobulin gene mutations in human melanoma cells: molecular characterization and implications for immune surveillance.

In recent years, the mechanisms underlying defects in the expression/function of human leukocyte antigen (HLA) class I antigens have been analyzed in an increasing number of melanoma cells, since these abnormalities are likely to have a negative impact on T-cell-based immunotherapy of melanoma. This article reviews the information about the molecular defects found in melanoma cell lines that do not express HLA class I antigens, following a concise description of the structure and assembly of HLA class I antigens. Distinct defects ranging from large deletions to point mutations in beta2-microglobulin genes have been found in melanoma cells. A mutation in an 8 base-pair CT repeat region of exon 1 has been found frequently in melanoma cell lines suggesting that this region of the beta2-microglobulin gene is a hot-spot for mutations. The effects of beta2-microglobulin mutations are mostly at the level of translation, emphasizing the importance of analyzing beta2-microglobulin expression at the protein level in melanoma lesions without detectable HLA class I antigen expression. Interestingly, many melanoma cell lines have additional defects that directly impact HLA class I antigen expression. Therefore, multiple mechanisms that affect the expression/function of HLA class I antigens appear to be available to melanoma cells to escape from immune recognition.

Exons↗
PubMed1996

Crystal structure of an anti-anti-idiotype shows it to be self-complementary.

The structure of the Fab fragment of the mouse anti-anti-idiotypic monoclonal antibody (mAb) GH1002 was solved by X-ray crystallography. mAb GH1002 was elicited with the syngeneic anti-idiotype mAb MK2-23 which mimics the determinant defined by anti-human high molecular weight-melanoma associated antigen (HMW-MAA) mAb 763.74. The Fab fragments of mAb GH1002 exist in the crystal as dimers related by crystallographic 2-fold axes. The interface between dyad-related Fab fragments is formed primarily by interaction of the hypervariable loops of one with the other. The self-interaction of Fab fragments of anti-anti-idiotypic mAb GH1002 through their combining sites is extremely tight and intricate, closely resembling that observed in structures of id-anti-id complexes, and comparable in terms of total contact area, charge complementarity, and number of hydrogen bonds. The self-complementarity of the antibody observed here could be coincidental and thus reflect some non-specific binding capability. It might, on the other hand, be immunologically relevant and exemplify a certain degree of evolved self complementarity characteristic of antibodies participating in idiotypic cascades.

Amino Acid Sequence↗
PubMed1996

Melanoma-associated hypopigmentation: where are the antibodies?

Antibodies to the B16 melanoma cell line and to tyrosinase have been recently defined in our laboratory in sera of patients with vitiligo, melanoma, melanoma-associated hypopigmentation (MAH), and in healthy subjects. The antibody titers in each subject were measured by enzyme-linked immunosorbent assay, were compared with the mean optical density (OD) of the control group, and were expressed as relative OD. The titers of anti-B16 antibodies (relative OD +/- standard error) were 1.000 (0.058) in the controls, 1.025 (0.077) in patients with metastatic melanoma, 0.5862 (0.15) in MAH, 1.377 in surgery-induced MAH, 1.087 in vaccination with anti-idiotypic antibodies, and 2.098 (0.15) in autoimmune vitiligo. The titers in vitiligo were significantly higher (p < 0.0001) than in MAH or in healthy controls. Antityrosinase antibodies were found in titers of 1.000 (0.1024) in the controls, 1.516 (0.225) in metastatic melanoma, 1.027 (0.180) in MAH, 1.075 in surgery-induced MAH, 2.308 in vaccination-induced MAH, and 4.536 in vitiligo. Differences were found between vitiligo and MAH (p = 0.008), surgery-induced MAH (p = 0.009), vaccination-induced MAH (p = 0.059), and healthy subjects (p < 0.0001). The results of this study point to the cross-antigenicity between melanocytes and melanoma cells, and to participation of antibodies against melanoma-associated membrane antigens in the mechanism leading to the development of MAH in patients with melanoma.

Adult↗
PubMed1996

Molecular and functional phenotypes of melanoma cells with abnormalities in HLA class I antigen expression.

Analysis of melanoma cell lines with abnormalities in HLA Class I antigen expression has identified two serological phenotypes caused by distinct molecular defects. One is characterized by lack of HLA Class I antigen expression which is not induced by IFN-gamma or by incubation at 25 degrees C for 24 hrs. This phenotype reflects structural changes in the beta(2)m gene which interfere with its transcription and/or translation or result in the synthesis of a defective beta(2)-mu polypeptide unable to associate with HLA Class I heavy chains. The other phenotype manifests very low HLA Class I antigen expression which is enhanced by IFN-gamma or by incubation at 25 degrees C for 24 hrs. This phenotype reflects abnormalities in TAP heterodimer expression, which cause defects in stable assembly and intracellular transport of the HLA Class I antigen trimolecular complex. Loss of HLA Class I antigens renders melanoma cells resistant to lysis by HLA Class I antigen-restricted cytotoxic T cells which specifically recognize melanoma associated antigens. Therefore, abnormalities in HLA Class I antigen expression may have a negative impact on the outcome of T cell based immunotherapy. Characterization of the molecular defects underlying loss of HLA Class I antigens may suggest approaches to restore their expression. Inclusion of these approaches in the protocols of T cell based immunotherapy may improve its efficacy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Development and characterization of rabbit antisera to human MHC-linked transporters associated with antigen processing.

The limited availability of sera to human MHC-linked transporters associated with antigen processing (TAP) has hampered the analysis of the role of these molecules in the reduced HLA Class I antigen expression by normal and transformed cells. To overcome these limitations, anti-human TAP1 and anti-human TAP2 xenoantisera have been generated and characterized. To this end rabbits have been immunized with TAP1-specific or TAP2-specific peptides which correspond to nonhomologous, hydrophilic regions of each transporter subunit. The immunized rabbits developed high titer IgG antibodies which displayed specific reactivity with the immunizing peptides in ELISA. Both anti-TAP1 and anti-TAP2 antisera immunoprecipitated the 70-76 kDa TAP complex from TAP(+)-TAP2+ cell lines WALK and Colo 38, but precipitated no component from TAP(-)-TAP2- cell lines T2 and SK-MEL-19. Furthermore, in immunodepletion experiments anti-TAP1 and anti-TAP2 antisera removed the molecules recognized by each of them in a lymphoid cell extract. Lastly, in Western blotting assays anti-TAP1 and anti-TAP2 antisera reacted specifically with isolated TAP1 and TAP2, respectively. The latter results in conjunction with those of the immunodepletion experiments indicate that TAP1 and TAP2 are not detectable as isolated subunits in a cell extract and that TAP heterocomplex is the major, if not the only detectable molecular species in cells. Anti-TAP1 and anti-TAP2 antisera were evaluated in immunohistochemical staining of both frozen and formalin fixed sections of skin and primary malignant melanoma lesions. Both antisera stained the cytoplasm of keratinocytes in normal skin and of melanoma cells in malignant lesions. The antisera we have elicited with TAP1- and TAP2-specific peptides appear to be useful reagents to characterize the role of TAP in abnormalities of HLA Class I antigen expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

The vitronectin receptor alpha-V beta-3, contrary to ICAM-1, is not modulated by interferon-gamma and tumour necrosis factor-alpha on melanoma cell lines.

A correlation was recently shown between expression of the vitronectin receptor (VnR) and the tumorigenic capacity of cultured human melanoma cell lines. On the other hand, modulation of VnR expression by interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) was observed on different non-melanoma cell lines. We tested IFN-gamma, TNF-alpha and interleukin-2 (IL-2), which are presumably released by infiltrating leukocytes in the melanoma lesional environment, on three melanoma cell lines. The VnR expression was assessed using FACS analysis and radioimmunolabelling. The VnR did not show any modulation after treatment with any of the cytokines tested. By contrast, the expression of the intercellular adhesion molecule-1 (ICAM-1), tested as control, on five melanoma cell lines, was greatly enhanced by IFN-gamma and TNF-alpha. Thus, some host cytokines may preferentially induce melanoma cells to express ICAM-1 (which can increase host cytotoxic response against melanoma), other than the VnR (which instead might contribute to melanoma metastasis).

Cytotoxicity, Immunologic↗
PubMed1996

Serum HLA class I antigen levels in allogeneic bone marrow transplantation: a possible marker of acute GVHD.

The levels of serum HLA class I antigens were determined at weekly intervals up to 5 weeks in 46 patients who had undergone allogeneic BMT. In patients with GVHD grade I or with GVHD grade I and fever of unknown origin (FUO), serum HLA class I antigen levels did not change during the observation period. In patients with GVHD grade II-IV serum HLA class I antigen level significantly increased in the week before the onset of GVHD, was maximal at the onset of GVHD and then persisted unchanged in the following 2 weeks. In patients with GVHD grade I or GVHD grade II-IV and infections whose onset coincided with that of acute GVHD a significant increase of serum HLA class I antigen level was found 2 weeks after the onset of the infectious episode. An increase of serum HLA class I antigen level was also found before the onset of repetitive GVHD grade II-IV episodes as well as during and after infectious episodes whose onset occurred after the onset of acute GVHD. The mean +/- s.d. concentrations of serum HLA class I antigens during GVHD grade II-IV episodes (9.4 +/- 3.4 micrograms/ml) and 2 weeks after the onset of infectious episodes (7.1 +/- 1.6 micrograms/ml) are significantly (P < 0.01 and P < 0.05, respectively) higher than that found 2 weeks before the onset of GVHD (3.0 +/- 0.5 micrograms/ml). The results of the present investigation suggest that measurement of serum HLA class I antigen level may be a possible marker to detect an acute GVHD following BMT.

Acute Disease↗
PubMed1995

Human high molecular weight-melanoma associated antigen mimicry by mouse anti-idiotypic MAb MK2-23. Immunohistochemical analysis of the reactivity of anti-anti-idiotypic MAb with surgically removed melanoma lesions.

The mouse anti-id MAb MK2-23 bears the internal image of the antigenic determinant defined by anti-HMW-MAA MAb 763.74. Active specific immunotherapy with anti-id MAb MK2-23 is associated with a statistically significant prolongation of survival in patients with malignant melanoma. Characterization of the immune response elicited by anti-id MAb MK2-23 may contribute to our understanding of the mechanisms underlying the apparent beneficial clinical effects of active specific immunotherapy with anti-id MAb MK2-23. Therefore, 8 HMW-MAA binding anti-anti-id MAbs elicited with MAb MK2-23 were characterized in their reactivity with a large panel of surgically removed benign and malignant tumors and of normal tissues. The 8 anti-anti-id MAbs displayed subtle differences in their immunoperoxidase staining of both benign and malignant lesions and of normal tissues. The diversity in the fine specificity of the 8 anti-anti-id MAbs is likely to reflect the few somatic mutations which occur in the amino-acid sequence of the variable regions of their heavy and light chains in the course of the immune response to MAb MK2-23. The reactivity patterns of the 8 anti-anti-id MAbs with the tissue substrates are similar, although not superimposable upon that of anti-HMW-MAA MAb 763.74 elicited with melanoma cells. This difference may reflect the imperfect mimicry by anti-id MAb MK2-23 of the antigenic determinant defined by anti-HMW-MAA MAb 763.74.

Animals↗