PubMed Health⌕ Search

Biomedical subjects

T Boon

Publications and source records attributed to T Boon.

At least 181 records · Page 10Linked to original sources
PubMed1985

Human T cell recognition of cloned HLA class I gene products expressed on DNA transfectants of mouse mastocytoma P815.

Cloned genes for human major histocompatibility complex (MHC) class I antigens were introduced by DNA-mediated gene transfer into a high-efficiency transfection recipient (HTR) cell line previously derived from mouse mastocytoma P815. Cell surface expression of HLA-A3, AW24 and CW3 gene products on P815 transfectants was demonstrated by radioimmune assay and by flow cytometry. The human MHC class I gene products were apparently expressed on P815 transfectants in a form recognized by human cytolytic T lymphocytes (CTL). Human CTL generated in unidirectional mixed lymphocyte culture against AW24+ donor lymphocytes clearly lysed P815-HLA-AW24+ transfectant target cells, but not untransfected P815(HTR) controls. Moreover, P815-HLA transfectants could stimulate in vitro a significant alloreactive human CTL response. Lysis of P815-HLA transfectant target cells by human CTL was inhibited by a monoclonal antibody directed against human MHC class I gene products. These mouse cell transfectants may be useful for the study of human T cell responses.

Animals↗
PubMed1985

A neomycin-resistant cell line for improved production of monoclonal antibodies to cell surface antigens.

Overgrowth of hybridomas by proliferating spleen T cells often hinders the production of monoclonal antibodies to certain antigens. To overcome this problem we derived neomycin-resistant fusion partner SP2 neoR.1 by transfection of SP2/0-Ag14 cells with plasmid pSVTK neo beta. Hybridomas obtained with SP2 neoR.1 grew optimally in the presence of the neomycin analog G418 at concentrations which blocked the proliferative response of T cells to mitogenic stimuli. The advantage of using SP2 neoR.1 and G418 under conditions where spleen cell proliferation occurs after fusion was demonstrated with hybridomas derived from a rat immunized with mouse helper T cell lines.

Animals↗
PubMed1985

Selection of highly transfectable variant from mouse mastocytoma P815.

A tk- cell line derived from mouse mastocytoma P815 was transfected with a plasmid carrying a thymidine kinase gene. The tk+ cells were obtained at a frequency of 10(-6). From some of these tk+ transfectants it was possible to select tk- cells with BrdU so that these cells could be submitted again to transfection with the thymidine kinase gene. By repeating cycles of transfection and tk+ selection followed by reverse selection with BrdU, it was possible to obtain a stable variant having a 100-fold increase in transfection efficiency. This HTR (high transfection) variant shows a high efficiency of transfection (10(-4)) for the neomycin-resistance gene as well as for the thymidine kinase gene.

Animals↗
PubMed1985

A limiting dilution assay for quantifying Leishmania major in tissues of infected mice.

A limiting dilution assay for the quantification of Leishmania major in infected mouse tissue was developed. The assay was found to be both sensitive and reliable, and, due to its design, could be scored either visually or following the incorporation of 3H-thymidine by the growing parasites. Results are presented in which the assay was employed to enumerate L. major in the tissues of susceptible (BALB/c) and resistant (CBA) mice at intervals after infection with L. major. It was found that parasites could be detected at the site of injection with L. major as early as 3 days after infection. By day 8, a substantial increase in the number of parasites at the lesion site had occurred in both strains of mice. Subsequently, whereas the number of parasites decreased in the lesions of CBA mice, their number steadily increased in the lesions of BALB/c mice. Parasites were detected in lymph nodes draining the lesion site in both BALB/c and CBA mice by 28 days after infection. Interestingly, a low number of L. major was found in the lymph nodes of CBA mice at 100 days after infection, a time when no parasites could be detected at the lesion site. Previous results from this laboratory have demonstrated that the adoptive transfer of L. major-specific L3T4-positive T-cell populations exacerbated cutaneous lesions induced by L. major in BALB/c mice. Experiments presented here indicate that the adoptive transfer of L. major-specific T-cells also exacerbated cutaneous leishmaniasis in CBA mice. Using the sensitive limiting dilution assay presently described, it was found that this unexpected exacerbative effect of L. major-specific T-cells on lesion development was accompanied by a substantial increase in the number of parasites in the lesions of the adoptively transferred mice.

Animals↗
PubMed1985

Tumour cell variants with increased immunogenicity obtained by mutagen treatment.

By treating mouse tumour cells in vitro with mutagens it is possible to obtain at high frequency variants that are rejected by normal syngeneic mice. Such variants have been designated 'tum-' to distinguish them from the original tumorigenic (tum+) cell lines. Most tum- variants appear to express new, individual antigens that can be defined in vivo by cross-protection experiments or in vitro by cytolytic T lymphocytes. The failure of tum- variants to form progressive tumours is apparently the result of an immune rejection response. Mice that have rejected a tum- variant clone are often protected against a subsequent challenge with the original tum+ cells. This was even demonstrated for two spontaneous mouse tumours for which no immunogenicity could be demonstrated otherwise. An analysis of 21 variants derived from mastocytoma P815 with cytolytic T lymphocyte populations showed that the repertoire of tum- antigens probably exceeds 50 specificities. No two variants have been found to express the same new antigen. Variant-specific long-term cytolytic T lymphocyte clones were isolated and used for the immunoselection of secondary antigen-loss variants that could then be analysed for the presence of previously undefined residual variant antigenic determinants. Somatic cell hybrids were prepared between different P815 tum- variants or between tum- variants and the original P815 cells. An analysis of the hybrids with variant-specific cytolytic T lymphocyte clones showed that the expression of tum- antigens was dominant. The intriguing paradox of the high frequency of tum- variants in populations of mutagen-treated tumour cells may require new genetic and immunological approaches before being understood.

Animals↗
PubMed1984

Partial purification of a membrane glycoprotein antigen by high-pressure size-exclusion chromatography without loss of antigenicity.

A high-pressure size exclusion chromatography system eluted with phosphate saline buffer containing 0.25% sodium deoxycholate has been developed that fractionates both soluble and membrane glycoproteins with good resolution and molecular weight versus elution time relationship. Using this system we fractionated membrane glycoproteins from a mutagenized mastocytoma cell that carries a strong transplantation antigen. After dialysis to remove the detergent, the fractions were tested for biological activity by an in vitro assay involving T-lymphocyte cell culture. Antigenic activity was found between 43 and 85 kdaltons. This demonstrates the efficiency of the system to resolve complex membrane protein samples without destroying their biological activity.

Animals↗
PubMed1984

Trypanosoma cruzi variants with reduced virulence obtained by mutagenesis.

Previous reports have indicated that by mutagen treatment of mouse tumour cells in vitro it is possible to obtain at high frequency stable tumour cell variants that fail to form tumours in syngeneic mice because of increased immunogenicity. By analogy with these tumour cell variants, we examined whether variants with reduced virulence could be obtained by mutagen treatment of trypomastigotes derived from a Trypanosoma cruzi strain that was adapted to culture and produced lethal infections in DBA/2 mice at a dose of 5 X 10(4) parasites. A very large frequency of T. cruzi clones were obtained that failed to provoke an acute lethal infection after injection of 5 X 10(5) parasites. Most of these variants with reduced virulence (vir-) multiplied actively in normal mice until day 8 after injection. After that time the parasitaemia decreased gradually. For most variants a low level of residual parasitaemia persisted for more than 100 days. Unlike the situation encountered with mouse tumour cell variants it was not possible to demonstrate the presence of new antigens on the T. cruzi vir- variants. However, these variants seemed to have acquired an increased immunogenicity since they provoked the rejection of virulent parasites injected concomitantly. Mice that had been immunized with living vir- clones were protected against a challenge with a virulent clone derived from the original parasite population.

Animals↗
PubMed1983

Protection against two spontaneous mouse leukemias conferred by immunogenic variants obtained by mutagenesis.

Two spontaneous mouse leukemias were adapted to culture. In agreement with most reported observations on spontaneous tumors, injection of irradiated cells of the malignant culture cell lines failed to protect mice against these leukemias. These cell lines were treated in vitro with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and stable immunogenic variants (tum-) were obtained, that failed to form progressive tumors in syngeneic CBA/Ht mice. Mice that had rejected tum- variants showed a significant degree of resistance to challenge not only with the original malignant cell line but also with the original transplantable tumor. No protection was observed against syngeneic tumor cells other than those of the parental tumor. These results indicate that these two spontaneous leukemias carry a specific transplantation antigen that can be the target of a rejection response by syngeneic mice. In confirmation of this, we found that lymphocytes of CBA/Ht mice that had rejected tum- variants could be restimulated in vitro so as to develop a cytolytic activity directed against an antigen that was specific for the original tumor cell line.

Animals↗
PubMed1983

Escape of mouse mastocytoma P815 after nearly complete rejection is due to antigen-loss variants rather than immunosuppression.

Even though mastocytoma P815 often undergoes a nearly complete rejection in syngeneic mice, the tumor cells almost always escape to form progressive tumors. We found that this was not due to the establishment of an immunosuppressed state because genetically marked P815 cells, that were injected in mice where tumor escape was occurring, were readily rejected. An analysis of escaping tumor cell populations with anti-P815 cytolytic T lymphocyte (CTL) clones showed the presence of stable resistant variants. Using antigen-loss variants found in escaping populations or selected in vitro with CTL clones, we were able to define four different tumor-associated antigenic specificities, each recognized by a specific CTL clone. One of these specificities was absent from all escaping tumor cells and another had been lost by some of them.

Animals↗
PubMed1983

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VI. Occasional escape from host rejection due to antigen-loss secondary variants.

After mutagenesis of mouse mastocytoma P815, it is possible to obtain variant (tum-) clones that are almost always rejected by syngeneic DBA/2 mice. Most tum- clones express new variant-specific antigens that can be detected by cytolytic T cells (CTL). Occasionally, mice injected with tum- variants eventually develop progressive tumors. Cells from these tumors were analyzed for antigen expression with variant-specific and P815-specific CTL clones. Out of 13 tumors examined, 11 were composed of cells that had clearly lost a variant-specific antigenic determinant. These results indicate that tum- variants induce a specific host rejection response which usually results in complete elimination of the variant cells, but that occasional antigen-loss constitutes an important mechanism of escape. The loss of a variant-specific antigenic determinant from one tum- clone that had escaped rejection allowed the detection of a residual variant-specific determinant. This was demonstrated by isolating a new CTL clone that lysed both the original tum- clone and its antigen-loss variant, but not other P815 targets. Thus, some complex transplantation antigens can be separated into independent determinants by using antigen-loss variants.

Animals↗
PubMed1983

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VII. Dominant expression of variant antigens in somatic cell hybrids.

After mutagenesis of mouse mastocytoma P815, it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic DBA/2 mice. Most of the variants express one or more new individual antigens specific for each variant, that are detectable in vitro by cytolytic T cells (CTL). Somatic hybrids were prepared either between tum- variants and the original P815 clone, or between different variants. Antigen expression of the hybrids was assessed by using long-term CTL clones that recognize specifically the new antigen present on the variants. Expression of tum- variant antigens behaved as a dominant trait in the hybrids. By submitting the somatic hybrids to selection with CTL clones, it was possible to obtain antigen-loss hybrid variants. The analyses of these antigen-loss variants showed that two variant-specific antigenic determinants associated with one of the variant fusion partners could be lost independently. Like the parental tum- variants, both the (tum+ X tum-) and (tum- X tum-) hybrids failed to form tumors in normal mice but formed tumors in irradiated mice.

Animals↗
PubMed1983

Increased frequency of immunogenic variants obtained by repeated mutagen treatment of mouse mastocytoma P815.

A previous report from this laboratory demonstrated that treatment of mouse mastocytoma P815 with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produces tumor cell variants that are unable to form tumors in syngeneic animals. We examined whether repeated mutagen treatment could increase the frequency of tum- variants above that obtained after a single treatment. This was found to occur with frequencies increasing from a few percent after 1 treatment to more than 90% after 8 treatments. Moreover, uncloned survivor populations obtained after 8 or more MNNG cycles that contained such a high proportion of tum- variants had a markedly decreased tumorigenicity for syngeneic mice. As reported for tum- variants obtained after 1 mutagen treatment, several tum- variants obtained after repeated treatments carried new variant-specific antigens that elicited a specific cytolytic T cell response. Some of these tum- antigens were found to consist of multiple determinants that could be lost independently. We observed that the resistance of the mutagenized populations to MNNG increased gradually with the number of mutagen treatments. In addition, some tum- variants obtained after 8 mutagen treatments showed a reduced sensitivity to mitomycin C.

Animals↗
PubMed1982

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. III. Clonal analysis of the syngeneic cytolytic T lymphocyte response.

The cytolytic T lymphocyte (CTL) response of syngeneic mice to antigenic variants obtained by mutagenesis of mastocytoma P815 was analyzed at the clonal level. Estimates of the frequency of CTL precursor cells in spleens from mice immunized with P815 variants ranged from 10(-4) to 2 X 10(-3). This frequency could be increased approximately 100 times by stimulating immune spleen cells in mass culture for 6-8 days with the immunizing variant. Specificity analysis of a large number of individual CTL clones demonstrated the existence of two distinct populations of CTL. Some CTL clones lysed exclusively the immunizing variant, while others lysed equally well all P815 targets, but not syngeneic tumor L1210. These results provide direct evidence for the existence of new, individual specificities on P815 variants in addition to a common antigen already present on the original P815 cells. This confirms that a variety of new antigens can be induced by mutagenesis on the same background cell. Stable, highly active CTL clones specific for either individual variant antigens or common P815 antigens could be maintained in long-term culture.

Animals↗
PubMed1982

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. IV. Analysis of variant-specific antigens by selection of antigen-loss variants with cytolytic T cell clones.

Mouse mastocytoma P815 tumor cell variants that express new individual antigens were subjected to immunoselection in vitro with specific cytolytic T lymphocyte (CTL) clones. From one variant (P35) a number of resistant clones were isolated after a single-step selection. These immunoselected clones (P35.iscA-) proved to have acquired a stable and complete resistance to lysis by the selecting CTL, but remained sensitive to lysis by a CTL clone that recognizes a common P815 antigen. However, when these P35.iscA- clones were used to stimulate in vitro spleen cells from mice immunized with variant P35, the cytolytic activity was significantly higher on P35 and P35.iscA- targets than on the original P815 clone (P1). This indicated that P35.iscA- cells, which had lost a P35-specific determinant (P35A), had nevertheless retained another P35-specific determinant (P35B). CTL clones directed against the residual P35B determinant were then isolated. When P35 cells were submitted to selection with anti-P35B CTL, resistant clones (P35.iscB-) were obtained that were still lysed by anti-P35A and anti-P815 CTL clones. Tum- variant P35 therefore carries at least two specific determinants that can be lost independently. These results show the possibility of using selection with CTL clones to separate different components of complex cell surface antigens. The correlation between antigen expression and tumorigenicity was also examined. Antigen-loss P35.iscA- clones were found to be more tumorigenic than variant P35 which has a greatly reduced tumorigenic capacity compared to the original P815 tumor. This correlation suggests that the P35 determinant that was no longer expressed by the P35.iscA- clones is recognized on the original P35 tum- variant as a tumor rejection antigen.

Animals↗
PubMed1982

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. V. H-2 associativity of variant-specific antigens.

By in vitro mutagenesis of mastocytoma P815, it is possible to obtain tumor cell variants that are rejected by syngeneic mice (tum-). Most of these variants carry new individual antigens and stimulate a specific cytolytic T cell (CTL) response in mixed leukocyte tumor cell culture (MLTC). The H-2 associativity of this response was examined for six different variants by measuring the inhibition of cell-mediated cytolysis by antibodies directed against products of the K or the D end of the H-2d complex. The lysis was either not inhibited (variants P91 and P116) or inhibited selectively by anti-Kd (variants P21, P32 and P198) or anti-Dd antibodies (variant P35). All these tum- variants expressed Kd and Dd antigens as measured by absorption of H-2 alloantisera. Long-term CTL clones can be obtained that are specific for individual tum- antigens. The pattern of H-2 associativity obtained with MLTC-derived CTL against four tum- variants was verified with CTL clones directed against the specific antigens of these variants. Concordant results were observed in all cases. In addition to CTL clones specific for tum- antigens, it is possible to isolate clones against a P815 tumor-associated antigen found on all P815 tum- variants. For these clones no clear associativity with either Kd or Dd products was found.

Animals↗
PubMed1982

Immunogenic variants obtained by mutagenesis of mouse Lewis lung carcinoma. Recognition of variant-specific antigens by cytolytic T lymphocytes.

By mutagenesis of a cell line derived from Lewis lung carcinoma (3LL), it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic mice. The possibility of obtaining a cytolytic T cell (CTL) response directed specifically against these tum- variants was examined. With the four variants that were analysed, a significant cytolytic activity was obtained with peritoneal cells from immune mice collected shortly after an intraperitoneal boost and also with spleen cells after a secondary stimulation in vitro. The CTL populations preferentially lysed the immunizing tum- variant, while also showing a cross-reactive lysis against the other variants and the original 3LL cells. Highly active CTL clones could be isolated from limiting dilution microcultures of these CTL populations. The clonal analysis clearly showed the existence of two distinct CTL populations, one directed exclusively against the immunizing variant and another that lysed all 3LL targets equally. This CTL specificity analysis therefore demonstrates directly the presence of new antigens on the 3LL tum- cell variants.

Animals↗