RESUMEN
The newly generated single-positive (SP) thymocytes undergo further maturation in the thymic medulla before their emigration to the periphery. The present study was undertaken to validate a developmental program we proposed for CD4SP medullary thymocytes and to explore the mechanisms regulating this process. During mouse ontogeny, the emergence of different subsets of CD4SP thymocytes followed a strict temporal order from SP1 to SP4. Parallel to the transition in surface phenotype, a steady increase in function was observed. As further evidence, purified SP1 cells were able to sequentially give rise to SP2, SP3, and SP4 cells in intrathymic adoptive transfer and in culture. Notably, the development of CD4SP cells in the medulla seemed to be critically dependent on a functionally intact medullary epithelial cell compartment because Relb and Aire deficiency were found to cause severe blockage at the transition from SP3 to SP4. Taken together, this work establishes an ontogenetically and functionally relevant maturation program for CD4SP thymocytes. Precise dissection of this program should facilitate further inquiry into the molecular mechanisms governing normal thymocyte development and its disturbance in pathological conditions.
Asunto(s)
Antígenos CD4/inmunología , Antígenos CD8/inmunología , Timo/citología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Timo/inmunología , Factores de Transcripción/genética , Proteína AIRERESUMEN
OBJECTIVE: To investigate the immune responses and protection from virus challenge, induced by the coinjection of IL-2cDNA with herpes simplex virus type 1 (HSV-1) glycoprotein-D (gD) DNA vaccine. METHODS: Two DNA vaccines (pgD and pIL-2) were constructed by inserting the gD gene and IL-2 cDNA into the eukaryotic expression vector pcDNA3.1, respectively. The BALB/c mice were inoculated intramuscularly three times at 2-week intervals. Two weeks after the final immunization, mice were bled for antibody assay and spleen cells were separated for Th cell proliferation and cytokine assays. Delayed type hypersensitivity (DTH) response was detected by the pinna-swelling test. Corneal protection under HSV-1 virus challenge was continuously observed with slit-lamp microscope. RESULTS: IL-2 cDNA coinjection remarkably enhanced the specific IgG2a level when compared with gD plasmid vaccination alone. Th cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by IL-2 cDNA coinjection. However, the production of IL-10 was inhibited. The DTH response was also enhanced by IL-2 coinjection. When the mice were challenged with HSV-1, the cornea epithelial lesions were significantly alleviated by IL-2 coinjection as compared with gD vaccination alone. CONCLUSION: IL-2 cDNA can enhance both the humoral and cellular immune responses, and thus increase the vaccine potency.
Asunto(s)
Hipersensibilidad Tardía/inmunología , Interleucina-2/genética , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genética , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Células COS , Proliferación Celular , Chlorocebus aethiops , ADN/genética , Femenino , Herpesvirus Humano 1/patogenicidad , Inmunización , Inmunoglobulina G/sangre , Interferón gamma/sangre , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Células TH1/citología , Transfección , Proteínas del Envoltorio Viral/biosíntesisRESUMEN
OBJECTIVE: To develop a therapeutic vaccine against human tumors associated with human papillomavirus type 16E6E7 (HPV16E6E7) which is modified from a Chinese patient of the cervical cancer which possessing the antigenicity and no transforming activity, and explore more active vaccine for inducing cellular immunity with mouse co-stimulatory molecular B7-1 gene. METHODS: The modified E6E7 gene expression plasmid pVR1012-fmE6E7 was constructed and transfected Cos-7 cells, and the E7 protein specific expression was testified by immunofluorescence assay. C57BL/6 mice were immunized intramuscularly with pVR1012-fmE6E7 alone or in combination with B7-1 gene expression plasmid (pcDNA3.1-B7-1). The activity of cytotoxic T lymphocytes (CTLs) was analyzed with 51Cr specific release assay and the specific antibody in sera was analyzed by indirect ELISA. HPV16 positive C57BL/6 tumor cells C3 were inoculated subcutaneously in the vaccinated mice to assay the growth of transplanted tumors. RESULTS: The specific CTLs and antibody from immunized mice were induced efficaciously by the E6E7 gene immunization, and co-administration of B7-1 gene could significantly enhanced the CTLs immune responses of fmE6E7, and protected 33% immunized mice against C3 tumor cells challenge. In contrast, all the mice immunized only with fmE6E7 gene developed transplanted tumors after C3 cells challenge. There was no difference in E7 specific antibody responses between mice immunized with the E6E7 gene only and co-administration with B7-1 gene. CONCLUSIONS: The modified E6E7 gene can be used as target gene for developing DNA vaccine, and B7-1 gene may represent an attractive adjuvant for enhancement of the specific cellular immune responses.
Asunto(s)
Antígeno B7-1/inmunología , Proteínas Oncogénicas Virales/genética , Proteínas Represoras , Neoplasias del Cuello Uterino/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antineoplásicos/inmunología , Antígeno B7-1/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Proteínas Oncogénicas Virales/inmunología , Proteínas E7 de Papillomavirus , Linfocitos T Citotóxicos/inmunología , Transfección , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
OBJECTIVE: The goal of this study was to construct a eukaryotic expression plasmid containing the gene encoding herpes simplex virus type I glycoprotein D (HSV-1, gD) and evaluate its utility for DNA immunization in mice. METHODS: The gD gene was amplified from viral DNA using PCR with EcoR I and BamH I restriction sites encoded on 5' and 3' ends, respectively. The PCR fragment was inserted into the transfer vector pGEM-T Easy. gD was then cut from this vector and inserted into the EcoR I and BamH I sites in the pcDNA3.1 at the multiple cloning sites (MCS). The recombinant plasmid, pcDNA3.1-gD1, was transfected into COS-7 cells using Lipofectamine according to the manufacture's instructions. The expression of the glycoprotein D was analyzed by immunoblotting of the cell lysates. 4-6 weeks old BALB/C mice were given two injections at tibia anterialis muscle, each containing 100 micrograms of plasmid DNA, on days 0 and 15. pcDNA3.1 was used as negative control. Blood samples were taken from all mice at weeks 0, 2, 4, and 6 after the first inoculation. Standard indirect ELISA was employed to evaluate the levels of specific total Ig in serum. RESULTS: The recombinant plasmid was confirmed with restriction digestion and sequencing to contain target gene segment and expressed in COS-7 cells in vitro shown by Western blotting. The pcDNA3.1-gD1 immunized group induced specific antibody response as compared to the negative control, and the titer was about 1:2000. CONCLUSIONS: The recombinant plasmid pcDNA3.1-gD1 is potential to be used as a candidate vaccine, for the treatment of HSV-1 infection.