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Immunosuppression through blockade of transferrin receptor: mechanisms
Immunosuppression Through Blockade of Transferrin Receptor: Mechanisms C. Rastellini, X. Li, M. Braun, M. Brown, L. Cicalese, and E. Benedetti
M
ONOCLONAL antibodies (MAb) against transferrin receptor (TfR) are effective in blocking cell proliferation because TfR is widely distributed on most proliferating and differentiated quiescent cells.1 TfR is involved in cellular growth and T-cell activation.2 In particular, the expression of TfR receptor is upregulated at the presence of antigen major histocompatibility complex (MHC). Blockade of TfR has been investigated in cancer immunotherapy.3 We have shown that, in a mismatched mouse combination, administration of TfR MAb for a short perioperative period can significantly prolong pancreatic islet graft survival (mean, 82 days) when compared to control animals (mean, 18 days, untreated or isotype-matched MAb-treated animals).4 Furthermore, we observed that the blockade of TfR with MAb through cytokine modulation induced an effective immunosuppression. This study follows up on the underlying mechanisms of islets graft prolongation attained by short perioperative administration of antiTfR MAb, particularly related to macrophage activity. MATERIALS AND METHODS The effect of anti-TfR MAb versus isotope control immunoglobulin G2a (50 g/mL on day 0) was evaluated in vitro by mixed lymphocyte reaction. Lymphocytes isolated from C57BL/6 mice were co-cultured with ␥-irradiated BALB/c cells and the supernatant was collected for interleukin (IL)-4 detection (enzyme-linked immunosorbent assay [ELISA]). Phenotypic analysis (CD4 and CD8) was also performed. Peritoneal macrophages were isolated from C57BL/6 mice and co-cultured with ␥-irradiated BALB/c islets. After 72 hours of culture, the supernatant was harvested for detection (ELISA) of IL-12 p70 and p40 levels to investigate the effect of blocking TfR on macrophages activity. Statistical analysis was performed using SPSS and the independent two-tailed Student’s t-test.
RESULTS AND CONCLUSION
IL-4 secreted by C57BL/6 lymphocytes was significantly reduced in the presence of anti-TfR MAb. IL-12 p70 was
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 33, 3279 (2001)
significantly downregulated by anti-TfR MAb. On the contrary, IL-12 p40 was significantly upregulated in the presence of anti-TfR MAb. We observed that CD8⫹ and CD4⫹ lymphocytes were respectively significantly suppressed (CD8⫹) and unmodified (CD4⫹) in the presence of antiTfR MAb. Our previous results provide unequivocal evidence for prolongation of islet allograft survival in MHCincongruent recipients that underwent short perioperative treatment with anti-TfR MAb. Blockade of TfR significantly affects IL-4 and IL-12 levels. In particular, blockade of TfR seems to interfere with macrophages activation through IL-12 p70 downregulation and IL-12 p40 upregulation. It is known that IL-12 p40 has an inhibitory effect on the interaction of IL-12 p70 and its receptor. Based on this, we observed that not only IL-12 p70 is directly downregulated but, furthermore, the upregulation of IL-12 p40 inhibits the binding of IL-12 p70 and its receptor with an overall inhibitory effect. The activity of anti-TfR MAb is represented by the significant suppression on CD8⫹ cells. Based on the in vivo and in vitro data, we strongly believe that treatment with anti-TfR MAb should be seriously considered for further studies as effective immunosuppressive strategy in transplantation. REFERENCES 1. May WS, Cuatrecasas P: J Membr Biol 88:205, 1985 2. Pattanapanyasat K, Hoy TG: Eur J Haematol 47:140, 1991 3. Kudoh S, Stanley J, et al: Int J Cancer 58:369, 1994 4. Rastellini C, Braun M, Li X, et al: Transplantation Proc 33:518, 2001 From the Section of Cellular Transplantation, Division of Transplantation, Department of Surgery, University of Illinois at Chicago, Chicago, Illinois. Address reprint requests to Cristiana Rastellini, MD, University of Illinois at Chicago, Division of Transplantation (MC 958), Room 402 Clinical Sciences Building, 840 South Wood, Chicago, Illinois 60612.
0041-1345/01/$–see front matter PII S0041-1345(01)02392-2 3279
M
ONOCLONAL antibodies (MAb) against transferrin receptor (TfR) are effective in blocking cell proliferation because TfR is widely distributed on most proliferating and differentiated quiescent cells.1 TfR is involved in cellular growth and T-cell activation.2 In particular, the expression of TfR receptor is upregulated at the presence of antigen major histocompatibility complex (MHC). Blockade of TfR has been investigated in cancer immunotherapy.3 We have shown that, in a mismatched mouse combination, administration of TfR MAb for a short perioperative period can significantly prolong pancreatic islet graft survival (mean, 82 days) when compared to control animals (mean, 18 days, untreated or isotype-matched MAb-treated animals).4 Furthermore, we observed that the blockade of TfR with MAb through cytokine modulation induced an effective immunosuppression. This study follows up on the underlying mechanisms of islets graft prolongation attained by short perioperative administration of antiTfR MAb, particularly related to macrophage activity. MATERIALS AND METHODS The effect of anti-TfR MAb versus isotope control immunoglobulin G2a (50 g/mL on day 0) was evaluated in vitro by mixed lymphocyte reaction. Lymphocytes isolated from C57BL/6 mice were co-cultured with ␥-irradiated BALB/c cells and the supernatant was collected for interleukin (IL)-4 detection (enzyme-linked immunosorbent assay [ELISA]). Phenotypic analysis (CD4 and CD8) was also performed. Peritoneal macrophages were isolated from C57BL/6 mice and co-cultured with ␥-irradiated BALB/c islets. After 72 hours of culture, the supernatant was harvested for detection (ELISA) of IL-12 p70 and p40 levels to investigate the effect of blocking TfR on macrophages activity. Statistical analysis was performed using SPSS and the independent two-tailed Student’s t-test.
RESULTS AND CONCLUSION
IL-4 secreted by C57BL/6 lymphocytes was significantly reduced in the presence of anti-TfR MAb. IL-12 p70 was
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 33, 3279 (2001)
significantly downregulated by anti-TfR MAb. On the contrary, IL-12 p40 was significantly upregulated in the presence of anti-TfR MAb. We observed that CD8⫹ and CD4⫹ lymphocytes were respectively significantly suppressed (CD8⫹) and unmodified (CD4⫹) in the presence of antiTfR MAb. Our previous results provide unequivocal evidence for prolongation of islet allograft survival in MHCincongruent recipients that underwent short perioperative treatment with anti-TfR MAb. Blockade of TfR significantly affects IL-4 and IL-12 levels. In particular, blockade of TfR seems to interfere with macrophages activation through IL-12 p70 downregulation and IL-12 p40 upregulation. It is known that IL-12 p40 has an inhibitory effect on the interaction of IL-12 p70 and its receptor. Based on this, we observed that not only IL-12 p70 is directly downregulated but, furthermore, the upregulation of IL-12 p40 inhibits the binding of IL-12 p70 and its receptor with an overall inhibitory effect. The activity of anti-TfR MAb is represented by the significant suppression on CD8⫹ cells. Based on the in vivo and in vitro data, we strongly believe that treatment with anti-TfR MAb should be seriously considered for further studies as effective immunosuppressive strategy in transplantation. REFERENCES 1. May WS, Cuatrecasas P: J Membr Biol 88:205, 1985 2. Pattanapanyasat K, Hoy TG: Eur J Haematol 47:140, 1991 3. Kudoh S, Stanley J, et al: Int J Cancer 58:369, 1994 4. Rastellini C, Braun M, Li X, et al: Transplantation Proc 33:518, 2001 From the Section of Cellular Transplantation, Division of Transplantation, Department of Surgery, University of Illinois at Chicago, Chicago, Illinois. Address reprint requests to Cristiana Rastellini, MD, University of Illinois at Chicago, Division of Transplantation (MC 958), Room 402 Clinical Sciences Building, 840 South Wood, Chicago, Illinois 60612.
0041-1345/01/$–see front matter PII S0041-1345(01)02392-2 3279