RESUMEN
In in vivo tolerance induction, the dose of tolerogen injected is generally linearly correlated to the length of tolerance induced. Small, medium and large doses are related to no, partial and long-term tolerance, respectively. However, even with injection of substantially large doses of tolerogen, the length of tolerance induced varies over a wide range. Most of the recipients can still reject donor grafts. In this study, it is shown that the linear dose-response can be altered into an all or nothing response in a H-Y antigen-specific TCR transgenic (Tg) mouse model. In thymectomized female Tg mice, injection of 3, 30 and 100 x 10(6) male spleen cells was correlated to no, partial and massive deletion of Tg (alpha T beta T) CD8 cells, respectively. When the thymectomized Tg mice were injected with 9 x 10(6) T cell-enriched (T+) male cells, one half of the recipients showed no deletion of alpha T beta T cells, and in the other half massive deletion occurred. In complete correlation with deletion, all male skin grafts were rejected in the undeleted group as PBS-injected controls, whereas with massive deletion they were indefinitely tolerized. Thus, partial deletion and partial tolerance can be avoided. Injection of 18 x 10(6) male T+ cells induced long-term tolerance in all the recipients. The all or none T cell deletion and long-term tolerance induction has not only significant implications in understanding the mechanism of peripheral tolerance induction, but also in tolerance induction in transplantation, gene therapy and the prevention and treatment of autoimmune diseases.
Asunto(s)
Epítopos de Linfocito T/inmunología , Antígeno H-Y/inmunología , Tolerancia Inmunológica/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Trasplante de Piel/inmunología , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Age-matched female C57BL/6 mice given injections i.v. of the syngeneic, poorly metastatic melanomas B16-F1 or JB/RH mount a measurable anti-melanoma antibody (Ab) response. The experiments described here examine the possibility of whether these Ab responses are in fact targeted against metastatically distinct subpopulations of melanoma. Reactivity of antisera and monoclonal antibodies (MoAb) developed by syngeneic immunization were examined by radioimmunoassay, flow cytometry, and Western blot analyses. Anti-melanoma antisera collected at various times after i.v. injection of melanoma cells were tested in radioimmunoassay against C57BL/6 melanoma cell lines and clones of well-characterized experimental metastatic activity. A strong inverse correlation between metastatic activity and Ab binding was observed. Furthermore, i.v. injection of poorly metastatic melanoma lines always resulted in strong Ab response, while highly metastatic clones did not. The relative distribution of Ab-reactive cells in melanoma populations was examined by flow cytometry. Again, the proportion of reactive cells in all melanoma cell lines and clones tested was inversely proportional to the degree of experimental metastatic activity. Hybridoma technology used to capture this selective Ab response yielded monoclonal reagents with distinct anti-melanoma specificity. Monoclonal antibody directed against nonmetastatic melanoma variants defined specific Mr 45,000 and 50,000 bands in Western blot analyses. Finally, melanoma cells nonreactive with the syngeneic Ab response were isolated after immunomagnetic bead plus magnet removal of the positive population. The experimental metastatic potential of these negatively selected cells was then compared with cells similarly fractionated with normal mouse serum or control MoAb. The results of these studies showed a clear increase in metastatic potential of anti-B16-F1 melanoma antiserum or MoAb-depleted cells. These results support the contention that host Ab responses may favor tumor progression and metastasis through selective Ab responses against the poorly metastatic population.
Asunto(s)
Formación de Anticuerpos , Melanoma Experimental/patología , Metástasis de la Neoplasia , Animales , Anticuerpos Monoclonales , Línea Celular , Citometría de Flujo , Variación Genética , Inmunización , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Bacillus amyloliquefaciens strain H is lysogenic for a large temperate phage we call H2. H2 has a polyhedral head 85 nm in diameter and a tail of about 17 x 434 nm. H2 lysogenizes Bacillus subtilis between the tyrA and metB genes, and gives specialized transduction of metB and, at lower frequencies, of ilvD and ilvA. The phage carries a thymidylate synthase gene and converts thymine auxotrophs of B. subtilis to prototrophy. The H2 genome is a linear DNA molecule about 129 kb in length. DNA extracted from phage particles grown in B. subtilis is not cut by the restriction endonucleases HaeIII, Fnu4HI, Bsp1286I, and BamHI; the latter enzyme is produced by B. amyloliquefaciens strain H. The prophage in lysogenic B. subtilis cells can be cut by these enzymes. We have isolated H2 mutants that carry the transposon Tn917, or a mutation resulting in clear-plaque morphology, or both.
Asunto(s)
Bacillus/metabolismo , Bacteriófagos/aislamiento & purificación , Bacteriófagos/genética , ADN Viral , Lisogenia , Transducción GenéticaRESUMEN
The restriction fragment patterns of two mutants forms of the temperate Bacillus subtilis bacteriophage SP beta have been examined. The DNA of a heat-inducible mutant, SP beta c2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SP beta c+ DNA. The DNA of a clear-plaque mutant, SP beta c1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SP beta c1 nor SP beta c2 DNA is cleaved by the endonuclease HaeIII.