OBJECTIVETo investigate the role of CD40 ligand (CD40L) in dendritic cells (DC) of CEA transgenic mice and to evaluate the specific cellular immunity induced by activated DC.

METHODSBone marrow cells of the CEA transgenic mice were used to generate immature dendritic cells under the condition of GM-CSF and IL-4. CD40L was added to activate dendritic cells into mature phenotype. Dendritic cells cancer vaccine was pulsed with CEA526-533 peptide which made the vaccine specific for cancer immunity. The immunophenotype molecules were identified by flow cytometry. The cytokines produced by cells were determined by ELISA. T cells proliferation was measured by (3)H-thymidine essays.

RESULTSImmunophenotype molecules expressions of CD40L-activated dendritic cells were significantly higher than those in control group. IL-12 secretion by CD40L-activated dendritic cells was (937.81+/-51.99) pg/10(6) DC, significantly higher than that in control group [(83.06+/-8.58) pg/10(6) DC, P<0.01]. CD8(+) T cells proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.05), and the secretion of IFN-gamma was(33.900+/-4.550) ng/L, significantly higher than that in control group [(5.226+/-0.460) ng/L, P<0.01]. Splenocytes proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.01), and the secretion of IFN-gamma was (69.802+/-11.407) ng/L, significantly higher than that in control group [(2.912+/-0.562) ng/L, P<0.01].

CONCLUSIONThe method of using CD40L to stimulate bone marrow-delivered dendritic cells promotes the maturation and activation of dendritic cells, which enhances the cellular immunity in CEA transgenic mice.

Animals ; CD40 Ligand ; immunology ; physiology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Immunity, Cellular ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic

Antiphospholipid Syndrome ; complications ; Atherosclerosis ; etiology ; B-Lymphocytes ; immunology ; CD11a Antigen ; genetics ; CD27 Ligand ; genetics ; CD40 Ligand ; genetics ; DNA Methylation ; Epigenesis, Genetic ; Humans ; Lupus Erythematosus, Systemic ; etiology ; genetics ; immunology

OBJECTIVEThe pathogenesis of bronchiolitis has not been fully identified. Immune function abnormality following virus infection may be associated with the pathogenesis. CD40 and CD40 ligand (CD40L) is a pair of co-stimulatory molecules in immunoreaction. They might play an important role in the development of bronchiolitis. This study aimed to investigate the expression of CD40 and CD40L in peripheral blood mononuclear cells (PBMCs) in children with bronchiolitis and explore their possible roles in the disease.

METHODSThirty children with bronchiolitis, 26 children with bronchopneumonia and 30 healthy children (control) were enrolled. Flow cytometry was used to detect CD40 and CD40L expression in PBMCs. Total serum IgE level was measured using ELISA.

RESULTSCompared with the control group, CD40L expression significantly increased in the bronchiolitis and bronchopneumonia groups (P< 0.05). The CD40L expression in the bronchiolitis group was significantly higher than that in the bronchopneumonia group (P< 0.05). A significantly increased CD40 expression was also found in the bronchiolitis group when compared with the bronchopneumonia and the control group (P< 0.01). Total serum IgE level in the bronchiolitis group was significantly higher than the bronchopneumonia and the control groups (P< 0.01). CD40 and CD40L expression was positively correlated with serum IgE level in the bronchiolitis group (r=0.607, r=0.819, respectively; P< 0.01).

CONCLUSIONSCD40 and CD40L expression in PBMCs and serum IgE level increased and there is a positive correlation between CD40 and CD40L expression and serum IgE level in children with bronchiolitis. Over-expression of CD40 and CD40L may play an important role in the development of bronchiolitis.

Bronchiolitis ; etiology ; immunology ; CD40 Antigens ; blood ; physiology ; CD40 Ligand ; blood ; physiology ; Female ; Humans ; Immunoglobulin E ; blood ; Infant ; Leukocytes, Mononuclear ; chemistry ; Male

OBJECTIVE: To investigate the expression of costimulatory molecules CD28/B7 and CD40/CD40L in T and B lymphocytes as well as its relations with total IgE (TIgE), eosinophil cationic protein (ECP) in serum and nasal allergic symptoms in patients with allergic rhinitis (AR). The effect of specific immunotherapy (SIT) on them were also investigated. METHOD: Thirty allergic allergic rhinitis patients were chosen as observation group, and 30 healthy patients as control group. Cytofluorometric analysis was used to compare the expression level of CD28/B7-1, B7-2 and CD40/CD40L on T cells and B cells in the two groups. The relationship between the CD28/ B7-1, B7-2 and CD40/CD40L expression level and serum Total IgE, ECP level were analyzed. RESULT: The expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells in allergic rhinitis patients were significantly higher than in the healthy, and serum level of TIgE has a positive relationship with the expression level of CD40L on T cells. ECP has a positive relationship with the expression level of B7-2 on B cells. The expression level of B7-1 showed no significant difference between the two groups. After specific immunotherapy for 6 months, the expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells were decreased in allergic rhinitis patients but still higher than in healthy. CONCLUSIONS The upregulated level of costimulatory molecules CD28/B7-2 and CD40/ CD40L on T cells and B cells may play an important role in the pathogenesis of allergic rhinitis, specific immunotherapy can downregulate the expression level of CD28/B7-2 and CD40/CD40L, and decrease the serum level of TIgE, it may be a possible mechanism in the treatment of allergic rhinitis.
Adult ; B-Lymphocytes ; immunology ; metabolism ; B7-2 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Case-Control Studies ; Desensitization, Immunologic ; Female ; Humans ; Immunoglobulin E ; blood ; Male ; Rhinitis, Allergic, Perennial ; blood ; immunology ; metabolism ; therapy ; T-Lymphocytes ; immunology ; metabolism

OBJECTIVETo explore the effect of CD40 blockade in suppressing acute rejection of renal graft in rats.

METHODSWith Wistar rats as the donor and male SD rats as the recipients, rat models of acute renal graft rejection was established. The rat models were divided into therapy group and control group, and in the former group, CD40 ligand (CD40L) monoclonal antibody was injected daily for 4 consecutive days starting on the next day following kidney transplantation. On day 5 after the transplantation, the renal graft was harvested for histological examination, and graft rejection was evaluated semiquantitatively.

RESULTSThe mean semiquantitative score of the renal graft was 0.63∓0.52 in the therapy group, significantly lower than that of the control group (3.72∓1.48, P<0.05).

CONCLUSIONCD40L monoclonal antibody can inhibit acute renal graft in rats.

Animals ; Antibodies, Monoclonal ; pharmacology ; therapeutic use ; CD40 Antigens ; antagonists & inhibitors ; immunology ; CD40 Ligand ; immunology ; Female ; Graft Rejection ; drug therapy ; prevention & control ; Graft Survival ; drug effects ; Kidney Transplantation ; adverse effects ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.
Skin Transplantation/*immunology/methods ; Mice, Inbred BALB C ; Mice ; Male ; Interleukin-2/*immunology ; Immunoconjugates/*administration & dosage ; Graft Survival/*immunology ; Drug Combinations ; CD40 Ligand/*immunology ; Bone Marrow Transplantation/*immunology/methods ; Antibodies/*administration & dosage/immunology ; Animals

To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.
Skin Transplantation/*immunology/methods ; Mice, Inbred BALB C ; Mice ; Male ; Interleukin-2/*immunology ; Immunoconjugates/*administration & dosage ; Graft Survival/*immunology ; Drug Combinations ; CD40 Ligand/*immunology ; Bone Marrow Transplantation/*immunology/methods ; Antibodies/*administration & dosage/immunology ; Animals

OBJECTIVETo investigate the effect and its mechanism of reducing graft-versus-host disease (GVHD) by in vitro blockade of CD(40)-CD(40)L pathway in vitro, the donor T lymphocytes cultured in vitro with anti-CD(40)L mAb were transfused in bone marrow transplantation (BMT) GVHD mouse model.

METHODSC57BL/6(H-2b) spleen T cells were isolated as responder cells, and BALB/c(H-2d) spleen cells as stimulator cells. They were cocultured with or without Anti-CD(40)L mAb as anti-CD(40)L mAb group and control group, respectively. At day 5, the mixed lymphocyte response (MLR)-culture cells mixed with bone marrow cells and transfused respectively into the TBI conditioned recipient mice. The mice were divided into two groups: group A, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR control group; group B, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR anti-CD(40)L mAb group. The GVHD incidence and hematopoietic reconstitution were observed. Peripheral blood sera and spleen cells of the recipients mice were harvested at scheduled time points for the measurement of cytokines and T cell immunophenotyping with flow cytometry.

RESULTSThe incidence of GVHD in group A was 100% (10/10), and in group B was 20% (2/10). The percentage of H-2D(b) positive cells in group B (n = 8) was (93.54 +/- 2.32)% at day 40 after transplantation. The levels of cytokines in serum from group B were significantly lower than those from group A (P < 0.05). The expressions of CD(4)(+), CD(8)(+), CD(4)(+)CD(25)(+), CD(8)(+)CD(25)(+), CD(4)(+)CD(69)(+), CD(8)(+)CD(69)(+) and CD(4)(+)CD(40)L(+) were lower in group B than in group A (P < 0.05). The expressions of CD(8)(+)CD(40)L(+) and CD(4)(+)CD(45)RA(+) were similar in the two groups (P > 0.05).

CONCLUSIONBlockade of CD(40)-CD(40)L interaction in vitro could induce immune tolerance in vivo, reduce aGVHD in aGVHD mice model and form chimerism, which was mediated by inhibiting the Th1 and Th2 cytokines production, inducing tolerance of CD(4)(+) and CD(8)(+) cells to alloantigens. The obstruction of T cells activation after tolerance happened mainly at the early and mature phase of T cells activation. These provided the experimental basis for the use of anti-CD(40)L mAb in the clinical transplantation to prevent aGVHD.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Bone Marrow Transplantation ; adverse effects ; CD40 Antigens ; physiology ; CD40 Ligand ; immunology ; physiology ; Graft vs Host Disease ; prevention & control ; Interleukin-10 ; blood ; Interleukin-4 ; blood ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVETo analyze the cloning result of CD40 mutant from RPMI8226 cells, a multiple myeloma (MM) cell line, and study the change of the expressions of costimulatory molecules and the apoptosis of RPMI8226 cells after activated with CD40.

METHODSCD40 gene mutant in RPMI8226 cell was detected by RT-PCR and DNA sequencing. The cell lines were cultured with sCD40L, L929/CD40L, soluble 5C11 (an anti-CD40 mAb) plate-bound 5C11 and their respective controls. Their growth curves, change of phenotypes and cell cycles were detected. The signalosome of CD40 on RPMI8226 cells were analyzed with laser scanning confocal microscope.

RESULTSThere was a single base substitution (TCA-->TTA) in the open reading frame of CD40 from RPMI8226 cells, resulting in the conversion of a amino acid (Ser124Leu). Only plate-bound antibody could inhibit RPMI8226 cell proliferation [(2.5 +/- 0.6) x 10(5) vs (7.8 +/- 1.2) x 10(5), P <0.05] and cause G1 arrested [(58.0 +/- 3.6)% vs (42.0 +/- 2.3)%, P <0.05]. muCD40 was translocated to CD40 signalosome while CD40 activated.

CONCLUSIONThe mutated CD40 in RPMI8226 cell might decrease its affinity to CD40L, leading to the disorder of CD40 signal.

Antibodies, Monoclonal ; pharmacology ; CD40 Antigens ; genetics ; immunology ; CD40 Ligand ; genetics ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Mutational Analysis ; Humans ; Multiple Myeloma ; genetics ; pathology ; Mutation ; Phenotype ; Transgenes

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice', and provide the evidences of acquired immune tolerance for allogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups: (1)CTLA4Ig+ anti-CD154 isotype control IgG; (2)anti-CD154 +CTLA4Ig isotype control IgG; (3)CTLA4Ig and anti-CD154; (4)isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control IgG. Each mouse in all groups received CTLA4Ig and anti-CD154 (or corresponding isotype control IgG) 500 µg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19(+)CD69(+)B cells, CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19(+)CD69(+)B cells were significantly increased in comparison with the normal group (P < 0.01) , whereas the numbers of cells were significantly decreased when blocking B7/CD28 or /and CD40/CD154 co-stimulatory signals (P < 0.01) . Blocking these 2 signals together displayed a synergistic effect (P < 0.01) . The central memory and effector T cells were defined as CD44(high)/CD62L(high) and CD44(high)/CD62L(low)/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signals together, displayed a synergistic effect (P < 0.01). Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluorescence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence intensity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group (P < 0.01) . It is concluded that blocking the B7/CD28 or/and CD40/CD154 co-stimulatory signal can inhibit the cellular and humoral immune function, whereas blocking these two signals together displays a synergistic effect.
Animals ; B7-1 Antigen ; metabolism ; Bone Marrow Transplantation ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Immune Tolerance ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Signal Transduction ; Transplantation, Homologous