Biomedical subjects

F Ronchese

Publications and source records attributed to F Ronchese.

At least 19 recordsLinked to original sources

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↗

PubMed1989

The contribution of mutant amino acids to alloantigenicity.

The I-Abm12 mutation has been used extensively to study the relationship between structure and function of murine class II major histocompatibility molecules. I-Abm12 differs from I-Ab by three amino acid replacements in the A beta chain, and the proposed structural model of the I-Abm12 molecule places these three amino acid substitutions along one of the alpha-helices where they may affect both antigen and TCR binding. Two of the substitutions, Ile----Phe67 and Thr----Lys71, are thought to point into the binding site, whereas the third substitution, Arg----Gln70, is thought to point up and hence, be available for binding to the TCR. These predicted orientations are consistent with serologic analysis of the bm12 molecule, which demonstrates that residue 70 is uniquely accessible to mAbs distinguishing I-Ab from I-Abm12. In this study we have determined the influence of each of these amino acid substitutions on the ability of the resulting molecules to stimulate a panel of I-Abm12 (allo) reactive T cell hybridomas. Our experiments indicate that reversion of the amino acid at position 70 from Gln (I-Abm12) to Arg (I-Ab) interferes with allorecognition by 33 of 35 I-Abm12-reactive hybridomas. On the other hand, many hybrids can tolerate amino acid substitutions at positions 67 or 71. Single amino acid substitutions at position 67, 70, or 71 are recognized by only a minority of I-Abm12-specific hybrids and usually the reactivity is greatly diminished. These data are most consistent with the idea that the amino acid at position 70 directly interacts with the TCR during allorecognition. The additional effects of residues 67 and 71 are consistent with a contribution by bound peptide to the allorecognition process.

Amino Acids↗

PubMed1989

Diminished antigen processing by endosomal acidification mutant antigen-presenting cells.

The role of acidified endosomes in Ag processing was investigated using mutant Chinese hamster ovary cells that express temperature sensitive defects in their acidification mechanism. These cells were transfected with MHC class II genes to convert them to APC. When such mutant cells were incubated at the nonpermissive temperature, losing early endosomal but not lysosomal acidification, their ability to process several native protein Ag was impaired. The nonpermissive temperature did not affect Ag processing by transfected wild type parental cells. Furthermore, T cells were stimulated normally under these conditions when the mutant cells presented antigenic peptide fragments, which do not require processing. The mutant cells were also not defective in the uptake and overall degradation of native antigen. The elimination of Ag processing by paraformaldehyde and chloroquine treatment of the Chinese hamster ovary cells indicates that these cells do not use a different process than physiologic APC. These results suggest that acidification of early endosomes is an important event in Ag processing.

Animals↗

PubMed1988

The molecular basis of antigen presentation.

We have used a multifactorial approach to investigate the relationship between the structure of class II major histocompatibility complex (MHC)-encoded cell surface molecules (Ia) and the recognition of Ia-bound peptide antigens by clonally distributed alpha beta heterodimeric T cell receptors (TCR). Four distinct parameters of Ia structure-function--1) control of Ia assembly and transport to the surface membrane; 2) serological reactivity with panels of monoclonal antibodies; 3) ability to present peptide antigens to T cells for functional activation; and 4) differential peptide binding independent of the TCR--have been measured. This has allowed assignment of allelically polymorphic subregions or individual residues to locations in a model class II molecular structure and attribution to these subregions and residues of specific functions such as control of alpha beta chain interaction and protein folding, antigenic peptide binding, or TCR binding. The results of these analyses have provided an internally consistent picture of the class II molecule that fits well with a hypothetical model for Ia derived by analogy from the recently solved HLA class I crystal structure. They have also given us the first clear definition of specific peptide binding residues within the general peptide binding region of Ia and have revealed an unexpected asymmetry in the structure-function relationships of the putative alpha and beta chain helical regions. Overall, the results of these studies indicate the critical importance of multi-parameter analysis in creating useful molecular models using non-chemical techniques. They also suggest that hypotheses about TCR-Ia interaction may have to take into account a significant asymmetry in the function of the two major polymorphic regions of histocompatibility molecules.

Animals↗

PubMed1987

Structure-function analysis of the Abm12 beta mutation using site-directed mutagenesis and DNA-mediated gene transfer.

The B6.C-H-2bm12 (bm12) mouse possesses a naturally occurring mutation in its class II MHC A beta gene. The three amino acid substitutions at positions 67, 70, and 71 that comprise this mutation lead to changes in both Ia expression and immune recognition of the resultant A beta A alpha molecule. The experiments reported here utilize a combination of oligonucleotide-mediated site-directed mutagenesis and DNA-mediated gene transfer to explore the roles played by each of the three mutant residues in these various phenotypic changes. A beta genes comprising all permutations of the residues distinguishing Ab beta from Abm12 beta were created and were individually co-transfected with Ab beta into mouse L cells. Sublines expressing high levels of membrane Ia were selected by preparative flow cytometry and were studied for reactivity with a panel of monoclonal anti-Ia antibodies, or for their ability to act as antigen-presenting cells (APC) for the stimulation of T cell hybridomas. During the generation of these transfectant lines, it was noted that expression of a high level of Abm12 beta Ab alpha was more difficult to achieve than a similar level of Ab beta Ab alpha. Northern blot analysis of specific A beta and A alpha mRNA levels in these various lines indicated that more class II mRNA, and presumably more A beta and A alpha chains, were required to achieve expression of Abm12 beta Ab alpha equal to that of Ab beta Ab alpha, suggesting that the previously noted reduction of Ia expression on cells from bm12 mice reflects a decreased ability of Abm12 beta Ab alpha chains to pair, or to reach the membrane. Staining of the panel of transfectants with monoclonal antibodies revealed that antibodies which did not distinguish Ab beta Ab alpha from Abm12 beta Ab alpha also reacted equally well with all molecules involving in vitro mutant A beta chains. Monoclonal antibodies reactive with Ab beta Ab alpha but not Abm12 beta Ab alpha were specific for an epitope primarily determined by the presence or absence of Arg 70 in Ab beta. In striking contrast, all three mutant positions were found to play crucial roles in T cell recognition, because all substitutions led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1986

I-A-restricted T cell antigen recognition. Analysis of the roles of A alpha and A beta using DNA-mediated gene transfer.

The contributions of A alpha and A beta chains, and of subregions of A beta, to Ia-restricted recognition of antigen by Th lymphocytes were analyzed using a panel of L cells transfected with various pairs of A alpha b,d, or k genes and recombinant or wild-type A beta b,d, or k genes. The A beta genes included all possible exchanges of the whole NH2-terminal (beta 1) domain or halves of the beta 1 domain among these three allelic A beta genes. The Ia+ L cells derived from such transfections were used as antigen-presenting cells with a 21 member panel of responding Ia-restricted T hybridoma cells of differing nominal antigen specificity and Ia-restriction. Special care was taken to account for quantitative variation in levels of Ia expression throughout the experiments. The results of this analysis reveal that (a) only 2 of the 21 Th cells recognized Ia molecules involving either a nonparental A alpha or a nonparental A beta chain, and in both cases the degeneracy extended to only one of the two other alleles tested. This suggests that allele specific contributions from both A alpha and A beta chains are important in restricted recognition for most, if not all I-A-restricted Th cells. (b) In no case did substitution of the A beta 2 domain from either of the alternative haplotypes lead to any functionally detectable effects, demonstrating that polymorphisms in the A beta 1 domain can entirely account for the restriction imposed on Th cell responses by the entire A beta chain. (c) For 90% of the cells tested, replacement of the NH2-terminal portion of the beta 1 domain with an allogeneic segment led to Ia molecules unable to elicit Th responses. Furthermore, of all the cells permissive of the substitution of one or other half of the beta 1 domain, only two permitted the substitution of sequence from both alternative haplotypes. Taken together, these data strongly suggest that antigen recognition by most, if not all, I-A-restricted Th cells involves contributions from both halves of the A beta 1 domain. These data suggest that the role of I-A molecules in restricted Th cell recognition of antigen depends on conformational determinants unique to a particular combination of polymorphic alpha and beta chains, and that multiple such sites exist on a single Ia molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Cordycepin reduces the sensitivity of BALB/Mo mouse lymphocytes to the induction of sister chromatid exchanges.

Lymphocytes from the spleen of BALB/Mo mice, which carry endogenous Moloney murine leukemia virus (M-MuLV), show in vitro frequencies of sister chromatid exchanges (SCEs) significantly higher than lymphocytes from control (M-MuLV free) BALB/c mice. In vitro treatment of lymphocytes with the antiviral antibiotic cordycepin (10 micrograms/ml) lowers the level of SCEs in BALB/Mo cells to the same value of BALB/c cells. M-MuLV yield is also markedly reduced in BALB/Mo lymphocytes cultured in the presence of cordycepin. The drug also abolishes the increased sensitivity of BALB/Mo lymphocytes to the induction of SCEs by mitomycin C (MMC) either in vitro (3 X 10(-8)/10(-7)M) or in vivo (0.3/3 mg/kg). Since cordycepin is known to inhibit poly(A)synthesis thus blocking RNA maturation, it is suggested that M-MuLV proviral integration is not per se the sole factor responsible for the more pronounced susceptibility of BALB/Mo lymphocytes to SCE induction, but most likely viral gene expression and amplification are needed for this effect to occur.

Animals↗

PubMed1985

Reversibility of lymphokine-induced NK-like activity in virus-specific cytotoxic T-lymphocyte clones.

A limiting dilution microculture system, supplemented with a source of interleukin-2 (IL-2), was employed to evaluate the frequency of Moloney-murine leukaemia/sarcoma virus (M-MuLV/M-MSV)-specific cytotoxic T-lymphocyte precursors (CTL-p) which also exhibited NK-like activity. Spleen cells, obtained from M-MuLV/M-MSV regressor mice, were restimulated in bulk secondary mixed leucocyte-tumour cell cultures (MLTC), and subsequently plated in a culture medium supplemented with two different supernatants (SN) produced following PMA-stimulation of the same EL-4 thymoma cell line. SN 20, obtained from the cell line maintained in vitro, contained IL-2 and only negligible amounts (less than 3 U/ml) of interferon (IFN), while SN 19, obtained after passage of the ascitic form of EL-4 thymoma in syngeneic mice, contained both IL-2 and IFN in high titres. The frequency of CTL-p specific for MBL-2 lymphoma cells was high and comparable in cultures supplemented with both SN (1/2 X 84 cells and 1/2 X 40 cells, respectively), while the frequency of CTL-p directed against NK-susceptible YAC-1 target cells was low in SN 20 (1/90 cells) and high in SN 19 (1/5 X 40 cells). An analysis of individual microcultures established at low cell dose (1 cell/well) indicated that specific and NK-like activity could be ascribed to the same precursor cells. Furthermore, using different long-term CTL clones, we observed that, after passage in SN 20, double-reactive clones gradually lose the capacity to lyse NK-susceptible targets, while most of MBL-2 specific clones acquired NK-like activity following a few passages in SN 19. Therefore, the induction of NK-like activity is reversible and may be modulated by soluble factors present in supernatant in which CTL clones are maintained. Double-reactive clones were unable to lyse NK-resistant allogeneic tumour cells or normal syngeneic blast cells. A few clones cross-reacting with H-2d alloantigens also exhibited NK-like activity when maintained in SN 19. The different pattern of CTL clone activity was associated with a morphological change in the clones themselves: the acquisition of double activity was accompanied by an increase in cell size and the appearance of numerous cytoplasmic granules. All CTL clones were phenotypically Thy-1+ and Lyt-2+ on indirect immunofluorescence and complement-dependent cytotoxicity investigation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Role of infectious virus expression and immune response in retrovirus-induced oncogenesis.

Some of the parameters involved in retrovirus-induced oncogenesis were analyzed in newborn mice injected with Moloney-murine leukemia virus (M-MuLV) as well as in adults who received the virus by the intraperitoneal (i.p.) or intrathymic (i.t.) route. The neonatally injected mice were permissive for both viral replication and virus-induced cell-surface antigen expression on thymus cells, peripheral T and B lymphocytes, and macrophages, whereas the M-MuLV was not present in the adult i.p. injected mice. Instead, in i.t. injected mice, the virus was expressed in thymus and peripheral T cells only, but was not detected in tail extracts as assessed by means of the UV-XC plaque assay. Lack of virus spread in adult-treated animals correlated with a prompt humoral and cellular immune response, whereas the highly viremic newborn inoculated mice showed negligible virus-specific antibody production and an extremely low frequency of splenic cytotoxic T lymphocyte precursors. Moreover, immune response in both groups of adult-treated mice efficiently prevented tumor induction by Moloney-murine sarcoma virus (M-MuSV), which has the same antigenic determinants as M-MuLV, its natural helper. In contrast, M-MuSV sarcomas grew progressively in newborn inoculated mice and killed the host. Finally, 80% of neonatally injected mice developed lymphomas, whereas all treated adults remained free of disease for more than 15 months. These findings imply that the immune response may, in fact, prevent retrovirus-induced oncogenesis through restriction of virus replication and/or destruction of virus-infected cells.

Age Factors↗

PubMed1984

Reduction in precursors of cytotoxic T lymphocytes and of cells with natural killer-like activity in spleens of cyclophosphamide-treated mice.

The effect of cyclophosphamide (CY) on precursors of cytotoxic T lymphocytes (CTL), specific for Moloney-murine leukemia virus (M-MuL V)- induced antigens, and on the precursors of cells having natural killer (NK)-like activity was studied by means of limiting dilution assay. Pre-treatment with a single CY dose of 100 mg/kg induced a marked reduction not only in the total spleen cell number but also in the frequencies of M-MuL V-specific CTL, and NK-like cell precursors. Maximal effect was obtained 2 days after CY injection, and a gradual recovery in both total spleen cell number and cytotoxic activity was achieved by day 12. These results confirm that CY exerts a strong but transient immunodepressive effect.

Animals↗

PubMed1984

Short- and long-term studies on chemical carcinogenesis in BALB/Mo mice.

To study the interactions between chemical carcinogens and oncogenic retroviruses, BALB/Mo mice which carry the Moloney murine leukemia virus (M-MuLV) as an endogenous virus, and conventional (M-MuLV-free) BALB/c mice, as well as their Bc1 (M-MuLV+ or M-MuLV-) hybrids were injected neonatally with a single dose of urethane. BALB/Mo and V+ Bc1 mice showed accelerated lymphoma development; similar results were obtained in BALB/Mo mice receiving one or two doses of urethane transplacentally. Lung adenomas developed with shorter latency and higher incidence in BALB/Mo mice given urethane at birth; however, significant differences in the incidence of lung adenomas in BALB/Mo mice were found only in two experiments. Additional short-term experiments were carried out to investigate the mechanism of the higher susceptibility to sister chromatid exchange induction observed in BALB/Mo lymphocytes. It was found that BALB/Mo spleen lymphocytes incubated with cordycepin, an antiviral antibiotic, with or without mitomycin C treatment, showed reduction in both M-MuLV synthesis and sister chromatid exchange frequency, and the latter values were similar to those seen in control cultures. These data suggest that the integration of M-MuLV proviral DNA into the host genome is per se not sufficient to increase the susceptibility to carcinogenic stimuli, but that other events, such as viral gene expression and amplification, are most likely required for the chemical-viral synergistic effect to occur.

Adenoma↗

PubMed1984

Virus-specific T cell response prevents lymphoma development in mice infected by intrathymic inoculation of Moloney leukaemia virus (M-MuLV).

Previous work on mice neonatally injected with Moloney-murine leukaemia virus (M-MuLV) had shown that T cell lymphoma development correlates with virus infection of lymphoreticular cells (T and B lymphocytes and macrophages) as well as with a lack of generation of virus-specific cytotoxic T lymphocytes (CTL) due to clonal deletion of CTL precursors. In the present report, viral antigen expression and T cell response in mice injected as adults with M-MuLV intrathymus (i.t.) was investigated. Only thymic and splenic T lymphocytes from these mice express virus-induced antigens since they were lysed by virus-specific CTL, and stained by anti-M-MuLV fluorescent serum. In addition, the percentage of M-MuLV-infected T cells increased with increasing post- inoculation times. However, these mice could mount a strong cellular immune response against M-MuLV-infected cells, as detected by massive mixed leucocyte tumour cell culture and by evaluation of virus- specific CTL precursor frequency. Finally, i.t. injected mice were not viraemic and did not develop lymphomas during an observation period of 12-15 months. These data, in contrast with the recent hypothesis that T cell lymphoma development depends on a chronic stimulation of virus-specific T lymphocytes, indicate that the cellular immune response is sufficient for prevention of neoplastic transformation, despite a persistent viral infection of the thymus and peripheral T lymphocytes.

Animals↗

PubMed1983

Sister chromatid exchanges and chromosomal aberrations induced by mitomycin C in mouse lymphocytes carrying a leukemogenic virus.

The induction of chromosomal damage (sister chromatid exchanges (SCEs), chromosomal aberrations, and micronuclei) in T lymphocytes from mouse spleen was analyzed after treatment in vivo with different concentrations of mitomycin C (MMC). Lymphocytes were derived from BALB/Mo mice, which carry an endogenous type C retrovirus (Moloney murine leukemia virus, M-MuLV), and from BALB/c mice (controls, M-MuLV-free). Chromosomal damage was determined in vitro on lymphocytes stimulated with concanavalin A (Con A) and incubated for two generation cycles with bromodeoxyuridine (BUdR). The baseline frequency of SCEs was significantly higher in untreated BALB/Mo than in BALB/c lymphocytes. The frequencies of SCEs were significantly increased by increasing doses of MMC in both BALB/c and BALB/Mo T lymphocytes. Treatment with a low dose of MMC (0.3 mg/kg) produced an additive effect on SCE frequency in BALB/Mo lymphocytes, which was gradually suppressed by increasing the MMC concentration (3-5 mg/kg). Indeed, the levels of SCEs became significantly lower in BALB/Mo than in BALB/c lymphocytes at the highest MMC concentration tested (10 mg/kg), indicating that a negative synergistic effect was eventually produced. Chromosomal aberrations (breaks and total aberrations) were significantly increased by the highest MMC doses (5-10 mg/kg) and were more frequent in BALB/Mo than in BALB/c lymphocytes at 10 mg/kg MMC. The frequencies of micronuclei were increased by all MMC doses and were significantly higher in BALB/Mo than in BALB/c lymphocytes at 10 mg/kg MMC. These results are referred to interferences of M-MuLV and MMC with the function of enzymes, such as DNA topoisomerases, involved in the mechanism of SCE production.

Animals↗

PubMed1977