Major histocompatibility complex (MHC) tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i.e. the ectodomain of H-2Db fused to a BirA substrate peptide (BSP), was constructed and overexpressed in E. coli BL21(DE3). Then, the denatured H-2Db-BSP was refolded in the presence of human beta2-microglobulin as well as the GP33-41 peptide (KAVYNFATC, KAV) of LCMV. The biotinylated H-2Db/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2Db KAV tetramer reagent was verified by detecting the CD8+ T cells specific for HCMV in KAV peptide vaccinated C57BL/6 mouse, with a mouse receiving subcutaneous injection of only adjuvant as negative control. The results showed that the tetramer positive rates were 0.27%, 0.11%, and 0.24% within the CD8+ T cell populations in the peripheral blood, draining lymph nodes, and spleen of vaccinated mouse, respectively. There was only very low background staining (< or = 0.01%) of those samples from the control mouse. Beside, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2Db tetramer will facilitate the study of the immune responses of antigen-specific CD8+ T cells in the experimental immunotherapy on the mice with H-2Db allele background.
Animals ; Antibody Specificity ; CD8-Positive T-Lymphocytes ; immunology ; H-2 Antigens ; genetics ; Histocompatibility Antigens Class I ; genetics ; Immunologic Techniques ; Lymphocytic choriomeningitis virus ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; T-Cell Antigen Receptor Specificity ; immunology

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.
Amino Acid Sequence ; Animals ; Enzyme-Linked Immunospot Assay ; methods ; Epitopes, T-Lymphocyte ; chemistry ; genetics ; immunology ; Female ; H-2 Antigens ; chemistry ; genetics ; immunology ; HIV Antigens ; chemistry ; genetics ; immunology ; HIV Infections ; immunology ; virology ; HIV-1 ; classification ; genetics ; immunology ; Histocompatibility Antigen H-2D ; Humans ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Peptide Mapping ; methods

In order to research the prophylactic effect of cyclosporine A (CSA) and mycophenolate mofetile (MMF) on GVHD in mice with H-2 haploidentical nonmyeloablative bone marrow transplantation, a murine model was established by using of C57BL/6J mouse as donor and (C57BL/6J x BALB/C) F(1) mouse as the recipient. The recipient mice were given CSA + MTX or CSA + MMF for prophylaxis of acute GVHD (aGVHD). The survival rate, hematopoietic recovery, and morbidity and mortality of aGVHD were observed for 50 days after transplantation. The results showed that typical aGVHD developed in the transplanted mice without prophylactic treatment during 22 to 25 days after transplantation. The morbidity of aGVHD was 75% (15/20), 40% (8/20) and 30% (6/20) and mortality was 100% (15/15), 62.5% (5/8) and 50% (3/6) respectively in unprophylactic group (control), CSA + MTX and CSA + MMF groups. In conclusion, CSA and MTX reduce the morbidity and mortality of aGVHD in mice with haploidentical nonmyeloablative bone marrow transplantation, and the effect of CSA + MMF is better than that of CSA + MTX.
Animals ; Bone Marrow Transplantation ; immunology ; Cyclosporine ; therapeutic use ; Graft vs Host Disease ; mortality ; prevention & control ; H-2 Antigens ; genetics ; Hematopoiesis ; Immunosuppressive Agents ; therapeutic use ; Male ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Mycophenolic Acid ; analogs & derivatives ; therapeutic use ; Transplantation Chimera

OBJECTIVEThe purposes of this study were to clone and sequence the major histocompatibility complex type I (MHC I) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy.

METHODSThe 1.4 kb full-length fragment of H-2Kk gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E. coli JM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2Kk cDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2Kk cDNA was determined by using Sanger's method.

RESULTSThe sequences of 615 mouse H-2Kk cDNA were 99% similar with those of H-2Kk cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other.

CONCLUSIONThe authors cloned the MHC I molecular antigen recognizing gene (H-2Kk) of 615 mice successfully and got the functional gene of MHC I.

Animals ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genes ; genetics ; Genes, MHC Class I ; genetics ; Genetic Therapy ; H-2 Antigens ; genetics ; Mice ; Mice, Inbred C57BL ; Point Mutation ; Sequence Analysis, DNA ; Transgenes

To explore the feasibility of nonmyeloablative conditioning regimens, hematopoietic reconstitution, chimera level and the occurrence of GVHD after nonmyeloablative allogeneic stem cell transplantation in H-2 haploidentical mice, CB6F1 mice were used as the recipient and were divided into 3 groups, mice were pretreated five days before transplantation. Group A was pretreated with myeloablative conditioning regimens (TBI with 10.5 Gy), group B was pretreated by TBI (2 Gy) + Ara-C + Cy and group C-TBI (2 Gy) + Ara-C + CY + Flu, respectively. For all recipient mice, the prevention of GVHD was not given, and 2 x 10(7) bone marrow cells mixed 1 x 10(7) spleen cells from C57BL/6 mice were injected through tail vein on day 0, and then hematopoietic recovery, engraftment and GVHD of recipients were observed. The results of chimera detection after transplantation showed that the engraftment of group A remained full donor chimerism, and engraftments of group B and group C were associated with mixed chimerism or full donor chimerism, but the chimerism of group B remained below 80% and tended to decrease after 50 days whereas chimerism of group C was above 80% (chimerism close to or being full donor type) and preserved even after 50 days. GVHD occurred in all the recipient mice due to that prevention was not given, wherein the occurrence and death rate of GVHD in group A was obviously higher than that of group B and group C (P <0.01), but there was no statistical difference between group B and group C. In conclusion, the nonmyeloablative conditioning regimens mainly based on fludarabine can form stable and lasting engraftment in the body of recipients. The mixed chimerism established in recipients induce tolerance of transplantation and decrease or avoid the occurrence of GVHD.
Animals ; Female ; Graft vs Host Disease ; prevention & control ; H-2 Antigens ; genetics ; Haplotypes ; Hematopoiesis ; Hematopoietic Stem Cell Transplantation ; mortality ; Male ; Mice ; Mice, Inbred C57BL ; Transplantation Chimera ; Transplantation, Homologous ; Vidarabine ; analogs & derivatives ; pharmacology

Using transplantable erythroblastic leukemia cells of EL9611(H-2d), the cells were inoculated to CB6F(1)(H-2d/b) generation of BALB/c x C57BL/6 mouse, the biological characterization of erythroblastic leukemia in haploidentical mouse was studied, that provides an experimental model for the study of graft-versus leukemia (GVL) with bone marrow or stem cell transplantation. When 10(3) - 10(8) of the spleen cells of EL9611(H-2d) had been intravenously inoculated to CB6F(1) mouse, the erythroblastic leukemia cells were transplanted successively and the F(1) generation of erythroblastic leukemia model in mice was established with 100% incidence of erythroblastic leukemia. There was a linear relationship between the survival time and the number of leukemic cell. The survival time of the mice was (9.6 +/- 0.8) days when 10(6) cells were inoculated. If the CB6F(1) mouse was transplanted successively for four generations, the incidence was 100%. The main targets for the leukemic EL9611(H-2d) cells were liver, spleen and marrow. The reaction of the erythroblastic leukemia cells for hemoglobin staining was positive, while the peroxidase reaction was negative. These cells were sensitive to some chemotherapeutic drugs, such as cytosine arabinoside and cyclophosphamide. This study presents the convenience for the studies on the GVL with haplo-allogeneic transplantation, in the F(1) generation of erythroblastic leukemia model of the commonly-used CD57BL/6 x BALB/c mouse.
Animals ; Cell Division ; Disease Models, Animal ; H-2 Antigens ; analysis ; Leukemia, Erythroblastic, Acute ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Neoplasm Transplantation ; Survival Analysis ; Tumor Cells, Cultured

OBJECTIVETo investigate the influence of glucocorticoid on phenotype of thymic dendritic cells in mice and to investigate the protective effect of Yougui Pill (YGP) on it.

METHODSBALB/c mice allocated in the group A and B were treated respectively with 10 mg/kg hydrocortisone, alone and combined with 20.81 g/kg YGP. The control mice were treated with normal saline. The changes before and after treatment of I-A(d) and H-2K(d) antigen presentation molecules expression in CD11c(+) and CD45(+) thymic dendritic cells of mice were analyzed by flow cytometry assay, and the expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) mRNA in thymocytes were determined by RT-PCR as well.

RESULTSThe percentage of I-A(d+) and H-2K(d+) in CD11c(+) in Group A after treatment was 46.77 +/- 4.32% and 64.34 +/- 7.69% respectively, as compared with those in the control group (65.81 +/- 7.69% and 31.88 +/- 5.01%), the percentage of I-A(d+) was lower and that of H-2K(d+) was higher significantly (all P <0.01). Meantime, the expression of ICAM-1 and LFA-1 in thymocyte in Group A (30.11 +/- 2.51% and 30.40 +/- 3.77%) was significantly lower than that in the control group (46.35 +/- 3.34% and 47.28 +/- 2.91%) respectively (P <0.01). Changes in Group B showed that treated by hydrocortisone in combination with YGP, the above-mentioned hydrocortisone-induced changes could be obviously reversed, the outcome of CD11c(+) I-A(d+) was 54.19 +/- 5.08%, ICAM-1 33.97 +/- 2.04% and LFA-1 34.80 +/- 2.92%, the difference between the two treated groups in these indexes all showed statistical significance (P <0.05).

CONCLUSIONGlucocorticoidcan inhibit the expression of major histocompatibility complex class II antigen molecule, but promote the expression of major histocompatibility complex class I in CD11c(+) and CD45(+) dendritic cells, down-regulate ICAM-1 and LFA-1 transcription, while the tonifying yang recipe, YGP, has a dominant protective effect against the above actions of glucocorticoid.

Animals ; CD11c Antigen ; metabolism ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; H-2 Antigens ; metabolism ; Histocompatibility Antigens Class II ; metabolism ; Hydrocortisone ; toxicity ; Intercellular Adhesion Molecule-1 ; metabolism ; Leukocyte Common Antigens ; metabolism ; Lymphocyte Function-Associated Antigen-1 ; metabolism ; Mice ; Mice, Inbred BALB C ; Phenotype ; Protective Agents ; pharmacology ; Thymus Gland ; cytology ; drug effects ; immunology

In order to explore what role relative cytokines play in acute GVHD (aGVHD) of mice transplanted with H-2 haploidentical nonmyeloablative bone marrow, a murine model was established by using C57BL/6J mouse as donor and (C57BL/6J x BALB/C) F1 mouse as the recipient. The recipient mice were given CsA and mycophenolate mofetile (MMF) for prophylaxis of aGVHD. The expression levels of IL-2, INFgamma, IL-4 and IL-10 in the spleen were detected by semi-quantitative RT-PCR at different time points after transplantation. The results showed that the expression levels of these cytokines increased slightly in the group only received nonmyeloablative conditioning. The expression levels of IL-2 and INF-gamma elevated significantly after transplantation in group 2 (without aGVHD prophylaxis), peaked at day 21 and day 14 respectively, then dropped rapidly, returned to the basal levels at day 35. The study on kinetic pattern of expression of IL-2 and INF-gamma in group 3 (with aGVHD prophylaxis) gave a similar result to group 2, but the peak value of cytokine expression in group 3 was much lower than that in group 2. The expression of IL-4 in Group 2 and group 3 all peaked at day 14, but the peak value in group 3 was higher than that in group 2, and decreased slowly in group 3. The expression of IL-10 increased gradually after transplantation, peaked at day 14, decreased after day 21, elevated again until day 35. It is concluded that the expression levels of these cytokines in the spleen all increase after H-2 haploidentical nonmyeloablative bone marrow transplantation. CsA and MMF can reduce the incidence and severity of aGVHD by down-regulating the expression levels of IL-2 and INF-gamma.
Animals ; Bone Marrow Transplantation ; immunology ; methods ; Cyclosporine ; administration & dosage ; Cytokines ; genetics ; Gene Expression ; drug effects ; Graft vs Host Disease ; prevention & control ; H-2 Antigens ; genetics ; Interferon-gamma ; genetics ; Interleukin-2 ; genetics ; Kinetics ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mycophenolic Acid ; administration & dosage ; analogs & derivatives ; Reverse Transcriptase Polymerase Chain Reaction

In order to explore a new special and effective way to prevent graft versus host disease (GVHD) after allogenic bone marrow transplantation (allo-BMT), the stem cell antigen-1 (Sca-1) + early hematopoietic cells (EHC) from BALB/c mouse (H-2d) were introduced with exogenous mouse Fas ligand (mFasL) cDNA gene by the retrovirus-mediated gene transfer and expanded for one week, and then they were co-cultured with the spleen mononuclear cells (SMNC) from BAC mouse (H-2dxb) as one way mixed lymphocyte reaction (OWMLR). The cytotoxicity of treated BAC mouse SMNC against Na2 51CrO4 labeling SMNC from BALB/c mouse was observed. The bone marrow mononuclear cells (BMMNC) from BAC mouse treated by the above methods were transplanted into lethally-irradiated congenic BALB/c mice to observe the occurrence of GVHD. The results showed that the SMNC from BAC mouse after OWMLR with exogenous mFasL cDNA gene-transduced hematopoietic cells (HC) from BALB/c mouse in a ratio of 1 to 5 exhibited an obvious inhibition of the cytotoxicity against the BALB/c mouse spleen cells at different effector/target ratios as compared to the control group (P<0.01). The grade I GVHD or no GVHD and the 80% survival rate at day 60 post-BMT were observed in the BALB/c mouse receiving BAC mouse BMMNC treated with similar way, while the grade II - III GVHD and the 20% survival rate were noted in the control group (P<0.01). It is suggested that the attenuation of GVHD in allo-BMT recipient could be successfully achieved through FasL-Fas pathway in an H-2 haplotype disparate mouse combination.
Animals ; Bone Marrow Transplantation ; Fas Ligand Protein ; Female ; Graft vs Host Disease ; immunology ; therapy ; H-2 Antigens ; genetics ; Haplotypes ; Hematopoietic Stem Cells ; cytology ; immunology ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Rats ; Rats, Wistar ; Signal Transduction ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology ; Transfection ; fas Receptor ; immunology

The potential therapeutic benefit of introducing IFN-gamma and GM-CSF genes in combination with the HSVtk suicide gene into subcutaneously implanted CT26 tumor cells was compared with that from each treatment alone. Cells, unmodified or retrovirally transduced with HSVtk or IFN-gamma/GM-CSF genes, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSVtk gene, with intraperitoneal ganciclovir treatment, reduced tumor volume by 81% at locally inoculated tumor sites (p<0.01) and by 25% at distantly inoculated tumor sites (p=0.052). IFN-gamma/GM-CSF genes showed a 56% tumor volume reduction at local tumor sites (p<0.01) and 15% volume reduction at remote tumor sites, although this was not statistically significant. The combination of HSVtk (with GCV) and IFN-gamma/GM-CSF genes showed an 81% volume reduction at local tumor sites (p<0.01) and a 43% volume reduction at remote tumor sites (p<0.01). Thus, the combination of HSVtk and IFN-gamma/GM-CSF gene therapy produced greater therapeutic efficacy than either treatment alone.
Animals ; Cell Line ; Cell Line, Tumor ; Gene Therapy/*methods ; Genes, Transgenic, Suicide ; Granulocyte Macrophage Colony-Stimulating Factors, Recombinant/biosynthesis/*genetics/therapeutic use ; H-2 Antigens/metabolism ; Interferon-gamma, Recombinant/biosynthesis/*genetics/therapeutic use ; Male ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental/*therapy ; Research Support, Non-U.S. Gov't ; Simplexvirus/enzymology/genetics ; Thymidine Kinase/*genetics/therapeutic use ; Transduction, Genetic