Freezing of dendritic cells, generated from cryopreserved leukaphereses, does not influence their ability to induce antigen-specific immune responses or functionally react to maturation stimuli.

It is of practical clinical importance to be able to reinfuse into patients dendritic cells which have been previously frozen in aliquots. However, there are few studies comparing the function of fresh and frozen dendritic cells. We therefore decided to perform a systematic immunophenotypic and functional comparison of fresh and frozen dendritic cells. We chose to assess functional properties using proliferation tests and evaluating the preservation of specific antigens presentation in the context of MHC class II. Dendritic cells, generated from leukaphereses of normal volunteers, were loaded with proteins by a 2-h incubation at a protein concentration of 50 microg/ml, and were thereafter used fresh or after freeze-thawing in an IFNgamma Elispot assay. The IFNgamma release from antigen specific T cells was not affected by liquid nitrogen storage of pulsed immature dendritic cells. In the same way, the storage did not alter their stimulatory properties for antigen specific autologous T cells or for allogeneic CD8+ T lymphocytes in a proliferation assay. We also showed that freezing non-pulsed immature dendritic cells did not alter their capacity to capture, process and generate antigen-specific reactions once thawed, nor did it impair their capacity to acquire fully mature characteristics using CD40L and IFNgamma, with respect to immunophenotype and bioactive IL-12 secretion.

The experimental (in vitro) and clinical (in vivo) immunosuppressive effects of a rat IgG2b anti-human CD2 mAb, LO-CD2a/BTI-322.

BACKGROUND: CD2 is a cell surface glycoprotein expressed on most human T cells and natural killer (NK) cells, working as a cell adhesion and costimulatory molecule. The aim of this paper is to analyze the mechanism of action of a rat IgG2b anti-human CD2 monoclonal antibody (mAb) (LO-CD2a/BTI-322 mAb), which is a potent immunosuppressive agent and inducer of cell death. In vivo, this mAb is able to prevent or treat kidney allograft rejection. METHODS: The mechanisms by which the LO-CD2a/BTI-322 mAb is able to induce inhibition of cell activation and cell death were analyzed by mixed lymphocyte reactions and by flow cytometry. After in vivo treatment, levels of circulating mAb were measured by ELISA as well as anti-rat immunization and cytokine release. RESULTS: We show that the inhibition of cell activation induced by LO-CD2a/BTI-322 mAb after allogeneic or OKT3 stimulation is due to an Fcgamma receptor-dependent CD2 down-modulation and to T-cell depletion through an antibody-dependent cell-mediated cytotoxicity mechanism mediated by NK cells or activated monocytes. Peripheral T- and NK-cell depletion was observed after in vivo treatment with LO-CD2a/BTI322. Cytokine release (TNFalpha) was correlated with some side effects, but only after the first injection, and the effects were never severe or life threatening. CONCLUSION: The correlation between the in vitro and in vivo data suggests that T-cell depletion, especially of activated cells, and inhibition of cell activation after CD2 down-modulation are the main mechanisms of action of the LO-CD2a/BTI-322 mAb.