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In vitro and in vivo effects of an anti-CD45 monoclonal antibody in the rat
In Vitro and In Vivo Effects of an Anti-CD45 Monoclonal Antibody in the Rat S. Ko, H. Nakano, A. Deiwick, A. Dinkel, K. Wonigeit, and H.J. Schlitt
C
OMMON leukocyte antigen (CD45) is a glycoprotein expressed on the cell surface of all hematopoietic lineage cells. CD45 molecules exert costimulatory function for T-cell activation through the T-cell receptor with tyrosine phosphatase activity.1 It is known that blockade of costimulatory signals can induce anergy of alloreactive T cells.2 Thus, we have tried immunomodulation of CD45 using a new monoclonal antibody (MAb) against an epitope of rat CD45 molecule. The purpose of this study is to evaluate the immunomodulatory effects of this new MAb in vitro and in vivo.
Flow cytometry revealed that our anti-CD45 MAb bound to more than 99% of peripheral blood leukocytes and lymph node cells of LEW and LEW.1W rats. Effect of Anti-CD45 MAb in Lymphocyte Proliferation Assay
Nonspecific and allospecific lymphocyte proliferation in vitro were significantly enhanced by anti-CD45 MAb in a dose-dependent fashion. Heart Transplantation Model
MATERIALS AND METHODS Animals l
RESULTS Characterization of Anti-CD45 MAb
u
LEW (RT1 ) and LEW.1W (RT1 ) rats were obtained from Zentralinstitut fu ¨r Versuchstierzucht in Hannover, and reared under conventional SPF conditions at the institute’s facilities.
MAb Rat MAb with specificity for the RT7 epitope of rat CD45 molecules was generated and purified in our laboratory. The affinity of this anti-CD45 MAb was evaluated by flow cytometry.
Lymphocyte Proliferation Assay Lymph node cells were prepared from LEW.1W rats. These cells were stimulated by soluble anti-CD3 MAb, Con-A, or irradiated allogeneic lymph node cells of LEW rats. Anti-CD45 MAb was added to the culture medium at various concentrations. The effect of added anti-CD45 MAb on the proliferation of responder cells was evaluated.
Heart Transplantation Model Vascularized LEW heart grafts were transplanted heterotopically into LEW.1W recipients by a standard procedure. Recipients were pretreated with a single dose of anti-RT7 MAb intravenously 1 day before transplantation (n ⫽ 8). Recipients receiving no MAb treatment served as controls (n ⫽ 6). The function of the grafts was monitored by daily inspection and palpation. Rejection was determined by cessation of graft beating and confirmed by the histologic presence of a mononuclear cell infiltrate and myocyte necrosis on hematoxylin-eosin sections.
Treatment of recipients with anti-CD45 MAb on day ⫺1 induced indefinite heart allograft survival, whereas untreated recipients of the same strain combination acutely rejected allografts within 8 days after transplantation. Recipients treated with anti-CD45 MAb showed a transient and significant drop in number of peripheral blood leukocytes, especially in number of T cells. DISCUSSION
Anti-RT7 MAb enhanced lymphocyte proliferation initiated by nonspecific and allospecific (MLR) stimulation in vitro, while showing a depleting effect on hematopoietic cells in vivo. A single perioperative dose of anti-CD45 MAb induced long-term allograft acceptance in a rat heart transplantation model. Knechtle et al3 reported that strong depleting antibody anti-CD3 immunotoxin induced tolerFrom the First Department of Surgery, Nara Medical University, Kashihara, Japan (S.K., H.N.); and Klinik fu¨r Abdominal-und Transplantationschirurgie Medizinische Hochschule Hannover, Hannover, Germany (S.K., A.De., A.Di., K.W., H.J.S.). Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 265, Project B5, and in cooperation with project B9. S.K. was supported by a fellowship from the Alexander von Humboldt Stiftung. Dr Ko’s present affiliation is First Department of Surgery, Nara Medical University, Nara, Japan. Address reprint requests to Dr Hans J. Schlitt, Klinik fu¨r Abdominal-und Transplantationschirurgie Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, D-30623 Hannover, Germany.
0041-1345/99/$–see front matter PII S0041-1345(99)00221-3
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1946
Transplantation Proceedings, 31, 1946–1947 (1999)
1947
EFFECT OF AN ANTI-CD45 MAB
ance in a renal allograft model. Because our anti-CD45 MAb also has a strong depletional effect on T cells, the mechanism of long-term allograft acceptance in this study might be similar to that of tolerance induced by anti-CD3 immunotoxin. CD45 has a costimulatory function required for signal transduction through the T-cell receptor.1 Modulation of this costimulatory function with anti-CD45 MAb can induce anergy in donor-reactive T cells of the recipient, which might result in long-term allograft acceptance in this model. Lazarovits et al4 provided evidence of prevention of allograft rejection using anti-CD45RB antibody. They also suggested that blockade of costimulatory signal would play a role in prolonged allograft survival in their mouse transplantation model.
The present study revealed that even a single dose of anti-CD45 MAb can induce long-term heart allograft acceptance, while enhancing lymphocyte proliferation activity in vitro. The precise mechanisms of action of anti-CD45 MAb have yet to be studied.
REFERENCES 1. Ledbetter JA, Tonks NK, Fischer EH, et al: Proc Natl Acad Sci USA 85:8628, 1988 2. Schwartz RH: Science 248:1349, 1990 3. Knechtle SJ, Vargo D, Fechner J, et al: Transplantation 63:1, 1997 4. Lazarovits AI, Poppema S, Zhang Z, et al: Nature 380:717, 1996
C
OMMON leukocyte antigen (CD45) is a glycoprotein expressed on the cell surface of all hematopoietic lineage cells. CD45 molecules exert costimulatory function for T-cell activation through the T-cell receptor with tyrosine phosphatase activity.1 It is known that blockade of costimulatory signals can induce anergy of alloreactive T cells.2 Thus, we have tried immunomodulation of CD45 using a new monoclonal antibody (MAb) against an epitope of rat CD45 molecule. The purpose of this study is to evaluate the immunomodulatory effects of this new MAb in vitro and in vivo.
Flow cytometry revealed that our anti-CD45 MAb bound to more than 99% of peripheral blood leukocytes and lymph node cells of LEW and LEW.1W rats. Effect of Anti-CD45 MAb in Lymphocyte Proliferation Assay
Nonspecific and allospecific lymphocyte proliferation in vitro were significantly enhanced by anti-CD45 MAb in a dose-dependent fashion. Heart Transplantation Model
MATERIALS AND METHODS Animals l
RESULTS Characterization of Anti-CD45 MAb
u
LEW (RT1 ) and LEW.1W (RT1 ) rats were obtained from Zentralinstitut fu ¨r Versuchstierzucht in Hannover, and reared under conventional SPF conditions at the institute’s facilities.
MAb Rat MAb with specificity for the RT7 epitope of rat CD45 molecules was generated and purified in our laboratory. The affinity of this anti-CD45 MAb was evaluated by flow cytometry.
Lymphocyte Proliferation Assay Lymph node cells were prepared from LEW.1W rats. These cells were stimulated by soluble anti-CD3 MAb, Con-A, or irradiated allogeneic lymph node cells of LEW rats. Anti-CD45 MAb was added to the culture medium at various concentrations. The effect of added anti-CD45 MAb on the proliferation of responder cells was evaluated.
Heart Transplantation Model Vascularized LEW heart grafts were transplanted heterotopically into LEW.1W recipients by a standard procedure. Recipients were pretreated with a single dose of anti-RT7 MAb intravenously 1 day before transplantation (n ⫽ 8). Recipients receiving no MAb treatment served as controls (n ⫽ 6). The function of the grafts was monitored by daily inspection and palpation. Rejection was determined by cessation of graft beating and confirmed by the histologic presence of a mononuclear cell infiltrate and myocyte necrosis on hematoxylin-eosin sections.
Treatment of recipients with anti-CD45 MAb on day ⫺1 induced indefinite heart allograft survival, whereas untreated recipients of the same strain combination acutely rejected allografts within 8 days after transplantation. Recipients treated with anti-CD45 MAb showed a transient and significant drop in number of peripheral blood leukocytes, especially in number of T cells. DISCUSSION
Anti-RT7 MAb enhanced lymphocyte proliferation initiated by nonspecific and allospecific (MLR) stimulation in vitro, while showing a depleting effect on hematopoietic cells in vivo. A single perioperative dose of anti-CD45 MAb induced long-term allograft acceptance in a rat heart transplantation model. Knechtle et al3 reported that strong depleting antibody anti-CD3 immunotoxin induced tolerFrom the First Department of Surgery, Nara Medical University, Kashihara, Japan (S.K., H.N.); and Klinik fu¨r Abdominal-und Transplantationschirurgie Medizinische Hochschule Hannover, Hannover, Germany (S.K., A.De., A.Di., K.W., H.J.S.). Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 265, Project B5, and in cooperation with project B9. S.K. was supported by a fellowship from the Alexander von Humboldt Stiftung. Dr Ko’s present affiliation is First Department of Surgery, Nara Medical University, Nara, Japan. Address reprint requests to Dr Hans J. Schlitt, Klinik fu¨r Abdominal-und Transplantationschirurgie Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, D-30623 Hannover, Germany.
0041-1345/99/$–see front matter PII S0041-1345(99)00221-3
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1946
Transplantation Proceedings, 31, 1946–1947 (1999)
1947
EFFECT OF AN ANTI-CD45 MAB
ance in a renal allograft model. Because our anti-CD45 MAb also has a strong depletional effect on T cells, the mechanism of long-term allograft acceptance in this study might be similar to that of tolerance induced by anti-CD3 immunotoxin. CD45 has a costimulatory function required for signal transduction through the T-cell receptor.1 Modulation of this costimulatory function with anti-CD45 MAb can induce anergy in donor-reactive T cells of the recipient, which might result in long-term allograft acceptance in this model. Lazarovits et al4 provided evidence of prevention of allograft rejection using anti-CD45RB antibody. They also suggested that blockade of costimulatory signal would play a role in prolonged allograft survival in their mouse transplantation model.
The present study revealed that even a single dose of anti-CD45 MAb can induce long-term heart allograft acceptance, while enhancing lymphocyte proliferation activity in vitro. The precise mechanisms of action of anti-CD45 MAb have yet to be studied.
REFERENCES 1. Ledbetter JA, Tonks NK, Fischer EH, et al: Proc Natl Acad Sci USA 85:8628, 1988 2. Schwartz RH: Science 248:1349, 1990 3. Knechtle SJ, Vargo D, Fechner J, et al: Transplantation 63:1, 1997 4. Lazarovits AI, Poppema S, Zhang Z, et al: Nature 380:717, 1996