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L Racioppi

Publications and source records attributed to L Racioppi.

31 records · Page 2Linked to original sources
PubMed1995

Serum withdrawal induces apoptotic cell death in Ki-ras transformed but not in normal differentiated thyroid cells.

Thyroid cells transformed by the Kirsten-ras oncogene become tumorigenic in syngeneic animals. Their growth is no longer dependent on TSH but becomes dependent on serum. Combining morphological and biochemical evidence, we show that serum withdrawal induces apoptotic cell death in Kirsten and Harvey-ras transformed thyroid cell. On the other hand, neither serum nor TSH withdrawal induce apoptosis in differentiated FRTL-5 cells. The induction of apoptosis by serum withdrawal is rapid and not triggered at a specific phase of the cell cycle. We suggest that induction of apoptosis following growth factor deprivation is an additional important characteristic, besides TSH-independence for growth and dedifferentiation, of the thyroid transformed phenotype.

Animals↗
PubMed1995

Cell surface expression of major histocompatibility class I antigens is modulated by P-glycoprotein transporter.

P-glycoprotein (Mdr1), a member of the ABC superfamily, is a pump able to transport several compounds across plasma membranes. It displays a high level of similarity with the MHC-linked transporters TAP1 and TAP2 which are involved in the delivery of immunogenic peptides across the endoplasmic reticulum. In the present study we analyze the P-glycoprotein's ability to interfere with the biosynthetic pathway of the MHC class I molecules. Our results show that P-glycoprotein is involved in the modulation of the MHC class I expression in multidrug-resistant tumor cell lines, COS1 cells transfected with mdr1 gene, and human T lymphocytes. Epitope screening evokes the possibility that P-glycoprotein induces a modulation of the different MHC class I forms expressed on the cell surface. We propose that P-glycoprotein is involved in the transport of antigenic protein fragments from the cytosol into the endoplasmic reticulum. The suggested mechanism could be physiologically relevant in tissues displaying a high Mdr1 activity, where this transporter could contribute to the regulation of locoregional immune responses.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Peptide-major histocompatibility complex class II complexes with mixed agonist/antagonist properties provide evidence for ligand-related differences in T cell receptor-dependent intracellular signaling.

Clonal activation of CD4+ and CD8+ T lymphocytes depends on binding of peptide-major histocompatibility complex (MHC) molecule complexes by their alpha/beta receptors, eventually resulting in sufficient aggregation to initiate second messenger generation. The nature of intracellular signals resulting from such T cell receptor (TCR) occupancy is believed to be independent of the specific structure of the ligand being bound, and to vary quantitatively, not qualitatively, with the concentration of ligand offered and the affinity of the receptor for the peptide-MHC molecule complex. In contrast to the expectations of this model, the analysis of the response of a T helper type 1 clone to mutant E alpha E beta k molecules in the absence or presence of a peptide antigen revealed that peptide inhibited the interleukin 2 (IL-2) response to an otherwise allostimulatory mutant form of this MHC class II molecule. The inhibition was not due to competition for formation of alloantigen, it required TCR recognition of peptide-mutant MHC molecule complexes, and it decreased IL-2 production without affecting receptor-dependent IL-3, IL-2 receptor alpha, or size enlargement responses. This preferential reduction in IL-2 secretion could be correlated with the costimulatory signal dependence of this cytokine response, but could not be overcome by crosslinking the CD28 molecule on the T cell. These results define a new class of TCR ligands with mixed agonist/antagonist properties, and point to a ligand-related variation in the quality of clonotypic receptor signaling events or their integration with other signaling processes. It was also found that a single TCR ligand showed greatly different dose thresholds for the elicitation of distinct effector responses from a cloned T cell population. The observations that changes in ligand structure can result in qualitative alterations in the effects of receptor occupancy and that quantitative variations in ligand density can be translated into qualitative differences in T cell responses have important implications for models of intrathymic selection and control of the results of active immunization.

Amino Acid Sequence↗
PubMed1993

The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition.

Previous studies on the role of specific residues of the peptide or MHC molecule in Ag presentation have revealed the sensitivity of this complex system to even small changes in structure. In our study, we have analyzed the effect of amino acid substitution in a major CD4+ T cell determinant (T1) of HIV-1 gp160 on binding and recognition in the context of various E alpha E beta MHC class II molecules. Individual alanine substitutions at all but three positions had little or no negative effect on either MHC binding or recognition by a specific T hybridoma, whereas substitutions with larger side chains often diminished reactivity. A poly-alanine peptide containing only four of the original residues was an effective MHC class II binder and in vivo immunogen, although lacking the ability to stimulate the hybridoma. Replacement of a glutamic acid in T1 with alanine or a size-conservative, uncharged glutamine, but not a negatively charged aspartic acid produced a peptide at least 100-fold more potent than the parent peptide, indicating an inhibitory effect of the negative charge. Conversely, substitution of a glutamic acid for valine at position 29 in the floor of the peptide binding site of the E alpha E beta molecule decreased functional presentation of this peptide by more than 2 logs. However, these two effects of glutamic acid were not complementary and were mediated by distinct mechanisms, as the change in the peptide altered the extent of binding to class II, but the change in the MHC molecule decreased recognition without inhibiting peptide binding. Taken together, the data all suggest the conclusion that changes in side-chains of peptides and MHC molecules affect Ag presentation and T cell stimulation most often by introducing dominant negative or interfering groups that prevent or alter the pattern of binding events primarily mediated by a very limited number of other residues in the Ag or presenting molecule. These results have important implications for understanding the biochemistry of peptide-MHC-TCR interactions and for the possible design of vaccines both more potent and less subject to allele-specific limitations on immunogenicity.

Amino Acid Sequence↗
PubMed1993

HLA class II molecules transduce accessory signals affecting the CD3 but not the interleukin-2 activation pathway in T blasts.

MHC class II molecules play a central role in the control of the immune response, but their biologic function and mechanism of action on the surface of activated human T lymphocytes are not entirely understood. In our study, the functional role of HLA class II molecules in T-blast proliferation was investigated by analyzing in parallel the IL-2- and CD3-driven activation pathways. The results indicate that the cross-linking of class II and CD3 molecules significantly increased the CD3-mediated T-blast proliferation, while no effect was observed on the IL-2-driven cell activation. This phenomenon was not confined to either CD4+ or CD8+ subsets nor was specifically affected by CD45 triggering. Biochemical studies showed that signaling via MHC class II molecules in T blasts led to PKC membrane translocation and IP accumulation. The simultaneous triggering of CD3 and HLA class II molecules led to a synergistic effect on IP accumulation but did not increase the CD3-mediated PKC membrane translocation. Our data suggest that HLA class II molecules are involved in T-cell-T-cell interactions and can mediate accessory signals, affecting the T-lymphocyte activation state.

CD3 Complex↗
PubMed1991

The molecular basis of class II MHC allelic control of T cell responses.

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.

Animals↗
PubMed1991

HLA class II molecules on monocytes regulate T cell proliferation through physical interaction in the CD3 activation pathway.

HLA class II molecules are involved in the OKT3-induced T cell activation, since monoclonal antibodies (mAb) to monomorphic determinants of class II antigens are able to inhibit T cell proliferation. This effect involves several of the events leading to T cell activation and proliferation, i.e. interleukin (IL) 1, IL 6 and IL2 secretion and IL2 receptor expression. The main target of the inhibition is represented by monocytes, and the interference of anti-class II mAb in the direct interaction of monocytes with T cells is likely to play a relevant role in the inhibition mechanism. To test this hypothesis, we investigated in the present study the effect of anti-class II mAb on the proliferation of T cells stimulated with mAb OKT3 in the presence of paraformaldehyde-treated monocytes. We show that the inhibition of proliferation is specific and dose dependent, and seems to involve particular class II epitopes. Addition of fixed monocytes to inhibited T cell cultures restores proliferation to a moderate extent, only if monocytes are added within the first 12 h of culture, suggesting that class II antigens or spatially related molecules deliver signals concurrently with the mitogenic stimulus. The blocking capability of anti-class II mAb was not restricted to the CD4+ or the CD8+ T cell subsets, suggesting that, under inhibitory conditions, these mAb affect other structures on the T cell surface, relevant to the monocyte-T cell interaction.

Antibodies, Monoclonal↗
PubMed1991

NK and LAK susceptibility varies inversely with target cell MHC class I antigen expression in a rat epithelial tumour system.

Several cell clones derived from cell lines obtained from a rat thyroid carcinoma, induced by in vivo injection of the Kirsten murine sarcoma virus into thyroid gland, and from its spontaneous lung metastases were analysed for their major histocompatibility complex (MHC) class I antigen expression. The susceptibility to natural killer (NK) cell lysis of these clones, differing in their levels of MHC class I antigen expression, was determined and found to vary inversely with the target cell MHC level, confirming numerous reports of the literature. We then tried to localize the step of the multistage natural cytotoxic process, in which class I antigens could interfere, and tested first whether lymphokine (IL-2) activation of the killer (LAK) cells could overcome the differences in MHC class I expression of target cells. As this did not appear to be the case, we studied the binding step by either a cold target inhibition assay and a target binding assay and found that target cells expressing class I antigens show a lower competitive capacity for effector cells than targets not expressing such antigens, indicating that this interference may occur, at least in our system, in the binding step of the cytotoxic process.

Animals↗
PubMed1990

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level.

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.

Antibodies, Monoclonal↗
PubMed1990

Monoclonal antibody OKT3-induced T cell proliferation: differential role of HLA class II determinants expressed by T cells and monocytes.

Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role of the corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CR10-343 and Q5/6 with high inhibitory effect and MAb CR11-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR10-343 and CR12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect of the combination of MAb CR10-343 and Q5/6 and of the MAb CR11-462 and CR12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3-induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli.

Antibodies, Monoclonal↗
PubMed1988

The relationship of modulation of major histocompatibility complex class I antigens to retrovirus transformation in rat cell lines.

The expression of major histocompatibility complex (MHC) Class I antigens has been studied, by means of monoclonal antibodies directed against nonpolymorphic determinants of MHC Class I molecules, in two epithelial differentiated cell lines (FRTL-5 clone 2 and PC clone 3) and in one fibroblast cell line (FRT Fibro) of Fischer rat thyroid origin, before and after infection with various acute retroviruses carrying the v-ras-Ha, v-mos, v-src, polyoma middle T, and c-myc oncogenes. The results obtained indicate that a single virus does not produce identical changes in MHC Class I molecule expression in all tested lines, but a general increase occurs in lines derived from FRTL-5 clone 2 and a decrease occurs in lines derived from PC clone 3 and from FRT Fibro. Thus the modulation of expression seems to proceed always in the same direction in each cell line regardless of the infecting retrovirus and appears to involve posttranscriptional mechanisms, since no modification of expression of mRNA levels has been observed between normal and transformed cells. Only one line of PC clone 3 origin, transformed by the cooperation of two oncogenes (human c-myc and middle T), almost completely lost MHC Class I antigens on the cell surface and presented a significantly reduced synthesis of Class I mRNA.

Animals↗
PubMed1987

Expression of major histocompatibility complex class I antigens in normal and transformed rat thyroid epithelial cell lines.

Recent evidence suggests that the expression of abnormally high amounts of major histocompatibility complex (MHC) class I molecules may be a feature of at least some kinds of transformed cells. To investigate this aspect of neoplastic transformation we studied the expression of MHC class I antigens in an experimental model of normal, tumor-derived, and virus-transformed thyroid epithelial cell lines. The expression of MHC class I antigens has been studied by means of several monoclonal antibodies directed against either monomorphic or polymorphic epitopes and quantified by flow cytometry. Class I specific mRNA transcripts have been also analyzed by Northern blot hybridization, using a mouse genomic H-2 DNA probe. Our results indicate a modulation of MHC class I expression associated with loss of the differentiated phenotype and with transformation of thyroid epithelial cell lines. Undifferentiated cells in fact show a small quantity of these antigens, because acquisition of a fully differentiated phenotype is associated with an increase in their expression. Cell lines derived from thyroid tumors show reduced expression of MHC class I antigens, as compared to differentiated cells. Conversely, cells transformed in vitro by a retrovirus carrying the v-raski oncogene exhibit an increase in these antigens in comparison to their normal differentiated counterparts. Cells infected with a mutant virus able to transform cells only at the permissive temperature of 33 degrees C show a similar increased expression. After a shift to the nonpermissive temperature of 39 degrees C, infected cells, even those losing the transformed phenotype retain the same increased amount of MHC class I antigens. Our data suggest that the modulation of MHC class I antigen expression is strongly associated with transformation in thyroid epithelial cells.

Adenocarcinoma↗