David A Clark1, Gerard Chaouat. 1. Department of Medicine, McMaster University, Hamilton, Ontario, Canada. clarkd@mcmaster.ca
Abstract
PROBLEM: Prevention of spontaneous abortion by allogeneic mononuclear leukocyte immunotherapy has proven ineffective in the CBA x DBA/2 murine abortion model if the leukocytes are stored overnight before inoculation. The mechanism and generality of the phenomenon has not been elucidated. METHODS: As prevention of recurrent abortion in the CBA x DBA/2 model requires allogeneic BALB/c lymphoid cells bearing paternal antigens and the tolerance-signaling molecule CD200 (OX-2), we evaluated effects of cell storage on cell surface CD200 expression using flow cytometry of fresh or stored cells stained with monoclonal anti-CD200 antibody. Release of putative CD200 molecules into culture supernatant during storage was tested by the ability of supernatants to block binding of anti-CD200 to freshly isolated cells. Similar studies were done using human peripheral blood mononuclear leukocytes. Possible binding of soluble CD200 to immunoglobulin G (IgG) molecules in plasma as a basis for the anti-abortive effect of intravenous immunoglobulin G (IVIG) was tested using the standard peripheral blood lymphocyte (PBL) natural killer (NK) cell lysis of (51)Cr-labeled K562 cells and monoclonal anti-human CD200 antibodies. RESULTS: Loss of anti-abortive effect of BALB/c cells with overnight storage at 4 degrees C and blocking of protective effect of freshly isolated cells with anti-CD200 antibody was confirmed. Supernatants of stored cells acquired a low level of protective activity against abortion in the CBA x DBA/2 model. Cell surface CD200 was lost with overnight storage at 4 or 22 degrees C, and supernatants acquired the ability to block binding of anti-CD200 antibody to fresh cells. Similar results were obtained using human PBL. However, if cells were stored overnight in IgG containing plasma, binding was not blocked. Suppression of NK cell lysis by PBL was abrogated if anti-CD200 antibody was added to the assay. CONCLUSIONS: Loss of the tolerance signal CD200 from allogeneic cells occurs with storage overnight, and their ability to protect against abortion is lost. CD200 appears to be shed into the supernatant, and may associate with IgG molecules rendering IVIG suppressive.
PROBLEM: Prevention of spontaneous abortion by allogeneic mononuclear leukocyte immunotherapy has proven ineffective in the CBA x DBA/2murine abortion model if the leukocytes are stored overnight before inoculation. The mechanism and generality of the phenomenon has not been elucidated. METHODS: As prevention of recurrent abortion in the CBA x DBA/2 model requires allogeneic BALB/c lymphoid cells bearing paternal antigens and the tolerance-signaling molecule CD200 (OX-2), we evaluated effects of cell storage on cell surface CD200 expression using flow cytometry of fresh or stored cells stained with monoclonal anti-CD200 antibody. Release of putative CD200 molecules into culture supernatant during storage was tested by the ability of supernatants to block binding of anti-CD200 to freshly isolated cells. Similar studies were done using human peripheral blood mononuclear leukocytes. Possible binding of soluble CD200 to immunoglobulin G (IgG) molecules in plasma as a basis for the anti-abortive effect of intravenous immunoglobulin G (IVIG) was tested using the standard peripheral blood lymphocyte (PBL) natural killer (NK) cell lysis of (51)Cr-labeled K562 cells and monoclonal anti-humanCD200 antibodies. RESULTS: Loss of anti-abortive effect of BALB/c cells with overnight storage at 4 degrees C and blocking of protective effect of freshly isolated cells with anti-CD200 antibody was confirmed. Supernatants of stored cells acquired a low level of protective activity against abortion in the CBA x DBA/2 model. Cell surface CD200 was lost with overnight storage at 4 or 22 degrees C, and supernatants acquired the ability to block binding of anti-CD200 antibody to fresh cells. Similar results were obtained using human PBL. However, if cells were stored overnight in IgG containing plasma, binding was not blocked. Suppression of NK cell lysis by PBL was abrogated if anti-CD200 antibody was added to the assay. CONCLUSIONS: Loss of the tolerance signal CD200 from allogeneic cells occurs with storage overnight, and their ability to protect against abortion is lost. CD200 appears to be shed into the supernatant, and may associate with IgG molecules rendering IVIG suppressive.