A monoclonal antibody to the rat Crry/p65 antigen, a complement regulatory membrane protein, stimulates adhesion and proliferation of thymocytes

A murine monoclonal antibody (mAb), 3F10, was produced by fusion of spleen cells obtained from mice immunized with a rat cortical thymic epithelial cell line (R-TNC.1) stimulated with interferon-gamma and P3X myeloma cells. 3F10 recognized an antigen expressed both on thymocytes and non-lymphoid cells in the thymus. Flow cytometry showed that 3F10 stained more than 98% thymocytes and 90% R-TNC.1 cells. Immunoprecipitation and Western blot studies demonstrated that 3F10 reacted with molecules of 55000 and 65000 MW from both thymocyte and R-TNC.1 cell lysates. 3F10 recognized the same antigen on Chinese hamster ovary cells transfected with rat Crry as did 5I2 mAb, confirming the specificity of 3F10 mAb for the rat homologue of mouse Crry/p65, a membrane-bound complement regulatory protein. 3F10 mAb induced homotypic aggregation of thymocytes and exhibited an additive effect on the aggregation evoked by phorbol myristate acetate. The aggregation was dependent on active cell metabolism, intact cytoskeleton, divalent cations and activation of protein phosphatases 1 and 2A (as assessed by use of okadaic acid). In contrast, H-7, HA1004 and genistein partially inhibited, whereas staurosporine potentiated the aggregation of thymocytes triggered by 3F10. 3F10 mAb also stimulated binding of thymocytes to the R-TNC.1 line. Both homotypic and heterotypic adhesive interactions are mediated by leucocyte function-associated antigen-1 (LFA-1). In addition, 3F10 stimulated proliferation of thymocytes induced by suboptimal concentrations of concanavalin A. These data suggest that rat Crry/p65 might be involved in the regulation of both cell adhesion and activation of thymocytes. This is a novel, non-complement-dependent function of Crry/p65

The role of rat Crry, a complement regulatory protein, in proliferation of thymocytes

In our previous work we showed that 3F10 monoclonal antibody (mAb), which recognizes the rat complement receptor 1-related/gene protein y (Crry), induces homotipic aggregation of thymocytes. In this work we studied the effect of 3F10 mAb on proliferation of rat thymocytes stimulated with concanavalin A (ConA) or by cross-linking the T cell receptor (TCR) by anti-alphabetaTCR mAb (R73), in vitro, and the mechanisms involved in the process. Our results show that 3F10 mAb stimulates proliferation of total thymocytes triggered by suboptimal concentrations of ConA or TCR cross-linking, in a dose-dependent manner. Maximal stimulation was observed using 10 mug/ml and 20 mug/Ml of 3F10 mAb, respectively. The 3F10-induced stimulation of thymocytes proliferation in the presence of ConA, that was followed by increased production of interleukin-2 (IL-2), up-regulation of the expression of IL-2 receptor alpha (IL-2Ralpha) and was inhibited by anti-CD11a and anti-CD 18 mAbs. Purified thymocytes did not respond by proliferation to 3F10 mAb, either alone or in combination with R73 mAb or ConA. Proliferation of these cells was achieved only in the presence of OX-6(+) antigen-presenting cells (APC) and additional signals transmitted by TCR or ConA. These results suggest that Crry is involved in the LFA-1 dependent proliferation of thymocytes, a phenomenon that has not been recognized so far

Characterization of antigen-presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry

Aim To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. Methodology Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. Results Antigen-presenting cells (HLA-DR+ cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR+ CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR+ CD14(+)), dendritic cells (DC) (HLA-DR+ CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR+ CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR+ cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P lt 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P gt 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P lt 0.05). Conclusion Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells

Comparison of two different protocols for the induction of maturation of human dendritic cells in vitro.

BACKGROUND: Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. METHODS: MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1beta, IL-6, tumor necrosis factor alpha (TNFalpha), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. RESULTS: MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. CONCLUSION: The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone