Production and Characterization of New Anti-Human CD20 Monoclonal Antibody.

The B-cell CD20 antigen is one of the most reliable surface targets in immunotherapy of B lymphoma. In this project, we studied the production and characterization of a new monoclonal antibody against chimeric human CD20 extra loops (hCD20 exl). The results showed that clone C12H, IgG2/k isotype reacted with the antigen in ELISA and immunoblot. The Kd value was found to be 2×10(-9)M and flow cytometry results showed that 99.9% and 99.7% of the Daudi and Raji cells respectively were stained with C12H monoclonal antibody (mab) but not with Jurkat cell lines. It also effectively competed with Rituximab, thus, the staining of the Daudi and Raji cell lines was reduced to 55.9% and 40.5% of cells respectively. Based on the high affinity reaction of C12H mab and appropriate reactivity of C12H mab with the native antigen on the surface of Raji and Daudi cells in flow cytometry, it was concluded that development and evaluation of C12H mab could be a beneficial candidate for further application in genetically engineered monoclonal antibody.

Expression of activation-induced cytidine deaminase gene in B lymphocytes of patients with common variable immunodeficiency.

OBJECTIVE: Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by reduced serum level of IgG, IgA or IgM and recurrent bacterial infections. Class switch recombination (CSR) as a critical process in immunoglobulin production is defective in a group of CVID patients. Activation-induced cytidine deaminase (AID) protein is an important molecule involving CSR process. The aim of this study was to investigate the AID gene mRNA production in a group of CVID patients indicating possible role of this molecule in this disorder. METHODS: Peripheral blood mononuclear cells (PBMC) of 29 CVID patients and 21 healthy controls were isolated and stimulated by CD40L and IL-4 to induce AID gene expression. After 5 days AID gene mRNA production was investigated by real time polymerase chain reaction. FINDINGS: AID gene was expressed in all of the studied patients. However the mean density of extracted AID mRNA showed higher level in CVID patients (230.95±103.04 ng/ml) rather than controls (210.00±44.72 ng/ml; P=0.5). CVID cases with lower level of AID had decreased total level of IgE (P=0.04) and stimulated IgE production (P=0.02); while cases with increased level of AID presented higher level of IgA (P=0.04) and numbers of B cells (P=0.02) and autoimmune disease (P=0.02). CONCLUSION: Different levels of AID gene expression may have important roles in dysregulation of immune system and final clinical presentation in CVID patients. Therefore investigating the expression of AID gene can help in classifying CVID patients.

In vivo effects of calcitriol on phenotypic and functional properties of dendritic cells.

BACKGROUND: Dendritic cells (DCs) play a central role in the initiation and expansion of T cell mediated immune responses with potential immunotherapy application. The compounds which have the ability to induce immunomodulatory effects on DCs may be employed for the treatment of immunopathologic conditions such as autoimmune diseases. OBJECTIVE: The aim of this study was to investigate the in vivo effects of calcitriol (active form of vitamin D3) on DCs. METHODS: 0.1 microgram calcitriol was injected intra-peritoneally into C57BL/6 mice every other day within 3 weeks, and spleen DCs were extracted by magnetic beads. The phenotypic and functional properties of DCs were studied by flow cytometry and mixed lymphocyte reaction (MLR), respectively. RESULTS: The expression of CD86 and MHC II, as maturation markers and costimulatory molecules were significantly decreased (p=0.028 and p=0.047, respectively) while CD11b expression, as a marker of mice myeloid DCs which mostly induces Th2 cytokine profile, was significantly increased (p=0.011). Allogeneic T cell stimulation in MLR was also significantly inhibited in comparison with the control groups (p<0.05). CONCLUSION: Our data indicate that in vivo calcitriol administration inhibits maturation and activation of DCs in the same manner as in vitro conditions.

Experimental autoimmune encephalomyelitis (EAE) induced by antigen pulsed dendritic cells in the C57BL/6 mouse: influence of injection route.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) of the immune system, and are critically involved in initiation of immune responses in autoimmune diseases. They can modulate the nature of immune responses to stimulatory or tolerogenic fashion. Previous studies have demonstrated that the administration route of DCs is an important variable in eliciting anti-tumor immunity. In this study we used experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis to compare different protocols of DC delivery in autoimmunity or tolerance induction. Dendritic cells were generated from bone marrow cells of C57BL/6 mice by culturing in the presence of GM-CSF and IL-4 for 7 days, followed by 2 days culture with TNF-alpha. The obtained DCs were pulsed in vitro with myelin oligodendrocyte glycoprotein (MOG) peptide and injected (5 x 10(5) cells/mouse) via the intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route into female C57BL/6 mice. In some instances pertussis toxin was also injected zero and 48 hours after DC injection. After follow up of the mice pretreated in this way for 4 weeks, in the i.v. group in which no clinical signs of EAE occurred, the mice were immunized with MOG peptide for EAE induction via the common method and the results were compared with mice that were not pre-immunized. Only after three s.c. DC injections with pertussis toxin, the mice showed mild clinical signs of EAE, whereas mice given i.v. or i.p. injections with or without pertussis toxin failed to develop EAE after 4 weeks. Induction of EAE via the common method after three injections of TNF-alpha treated DCs, in i.v. injected groups showed no protection from EAE. It seems that several factors influence the tolerance versus immunity induction by DCs. Our results showed that the administration route of DCs is one of the pivotal factors in DC-based induction of autoimmune diseases.

PPD extract induces the maturation of human monocyte-derived dendritic cells.

Dendritic cells (DCs) are the most powerful antigen presenting cells, capable of inducing T-dependent immune responses even in naive T cells. DCs are of special interest as cellular adjuvants for immunity induction in clinical settings and several methods for their generation and maturation are recently under investigation. The present study was set out to define the effects of PPD (Purified Protein Derivative), a mycobacterial extract used in the tuberculin skin test, on in vitro differentiation and maturation of human monocyte derived dendritic cells. Immature DCs were prepared from the peripheral blood monocytes of healthy volunteers by culturing in a medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). The resultant cells were then stimulated with PPD extract and their properties such as cell morphology and the expression of key surface molecules were compared with tumor necrosis factor-alpha (TNF-alpha) stimulated immature DCs. Our results suggest that mycobacterial purified extract is as potent as TNF-alpha, a well-established DC stimulator, in induction of maturation in human monocyte derived DCs. We also ruled out the contribution of lipopolysaccharide (LPS) and beta-glucan contamination in maturation effect of PPD preparations. So, PPD as an examined safe material for in vivo consumption could be used to stimulate DC maturation in DC based immunotherapy protocols.

Kinetics of murine decidual dendritic cells.

Dendritic cells (DCs) are professional antigen presenting cells (APC) capable of induction of primary immune responses as well as immunologic tolerance. Myeloid and lymphoid subsets of murine DCs are able to shift cytokine responses of T cells toward Th2 and Th1 profiles respectively. Thus, DCs would be suitable candidates to mediate the balance of maternal immune responses to conception. We analyzed pregnancy-related variations in uterus and splenic DCs in a murine model. C57BL/6-mated Balb/c female mice with vaginal plugs were scarified at early, middle, and late pregnancy. Frozen sections of uterus and spleen at each stage of pregnancy were immunostained with CD11c- and MHC-II-specific antibodies. Two-color immunohistochemistry was also carried out using anti-CD11c and one of the antibodies against CD11b, CD8alpha, CD86, and DEC-205. Using morphometric analysis, the average density of DCs and relative percentage of myeloid (CD11c+, CD11b+) and lymphoid DCs (CD11c+, CD8a+) were determined at each stage. Our results showed that DCs are present throughout the pregnancy in decidua. The average density of decidual DCs at early pregnancy was significantly higher relative to middle and late gestation or to those of endometrial DCs of non-pregnant mice. Interestingly, the average density of decidual and splenic DCs, followed the same variations at different stages of pregnancy. The relative percentage of decidual lymphoid DCs (LDC) was significantly higher at mid-gestation when compared with other stages of pregnancy or non-pregnant mice. Inversely, the frequency of myeloid DCs (MDC) and the MDC/LDC ratio were statistically lower at the middle stage of pregnancy. A majority of decidual DCs expressed MHC-II and CD86. At early pregnancy, DCs were more concentrated subadjacent to the luminal epithelial layers, whereas at mid-or late gestation, DCs were randomly distributed in the stroma and around the epithelium. Mid-pregnancy period was a critical point with regard to splenic DCs kinetics, as both the average density of DCs and the frequency of MDCs decreased significantly when compared with early or late pregnancy, although the relative percentage of splenic LDCs did not change. Our data suggest that the balance of MDC and LDC is finely tuned throughout pregnancy, pointing an eminent immunoregulatory role of DCs in the maintenance of pregnancy.

Analysis of endometrial myeloid and lymphoid dendritic cells during mouse estrous cycle.

This study was performed to evaluate the frequency and localization of endometrial myeloid (CD11c(+) CD11b(+)) and lymphoid (CD11c(+) CD8alpha(+)) dendritic cells (DCs) at different stages of murine estrous cycle. To address the systemic effect of ovarian hormones fluctuations during estrous cycle, the same variables were studied in splenic DCs as well. Stages of the estrous cycle of Balb/c mice were determined by examination of vaginal smears. Frozen sections of uterus and spleen at each stage of estrous cycle were stained for CD11c and MHC-II. Two-color immunohistochemistry was also carried out using anti-CD11c with one of the antibodies against CD11b, CD8alpha, CD86, and DEC-205. The average density of DCs and relative percentage of myeloid and lymphoid DCs (MDCs and LDCs) were determined at each stage of estrous cycle by morphometric analysis. Our results showed that DCs were present throughout the estrous cycle in mice endometrium, but their frequency was highest at estrus and lowest at proestrus (P<0.005). The lymphoid subset of DCs was more prominent at estrus relative to those at other stages (P<0.005). Conversely, the relative percentage of myeloid DCs at estrus was significantly lower compared to other stages (P<0.005). Nearly all endometrial and splenic DCs expressed CD86 and MHC-II. At proestrus, and particularly at estrus, DCs were more concentrated subadjacent to the luminal and glandular epithelial layers with some scattered throughout the stroma whereas, at metestrus and diestrus, DCs were randomly distributed in stroma and around the glandular and luminal epithelial layers. The number and immunophenotype of splenic DCs were not statistically different between stages of estrous cycle. Our results suggest that endometrial but not splenic myeloid and lymphoid DCs are influenced by steroid hormones during estrous cycle.