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Prolonged graft survival induced by CTLA4IG-Gene transfection combined with FTY720 administration in rat heart grafting
Prolonged Graft Survival Induced by CTLA4IG-Gene Transfection Combined With FTY720 Administration in Rat Heart Grafting Y. Kita, X.-K. Li, N. Funeshima, S. Enosawa, A. Tamura, S. Hayashi, K. Suzuki, T. Kazui, H. Amemiya, and S. Suzuki
C
TLA4Ig is a recombinant fusion protein that contains an extracellular domain of human CTLA4 and an Fc portion of human IgG1 heavy chain and blocks the B7/ CD28-mediated costimulatory signal, resulting in inhibition of T-lymphocyte activation.1 FTY720 is a synthetic drug produced by chemical modification of ISP-1 purified from culture filtrates of Isaria sinclairii. A single administration of FTY720 resulted in a marked reduction of peripheral lymphocytes.2,3 This study was conducted to define the combination effect of CTLA4Ig-gene transfection and perioperative administration of FTY720 in rat heart allografting.
OX-39; PharMingen, San Diego, Calif). We also stained apoptotic cells by the DNA nick-end labeling (TUNEL) method.
Flow Cytometric Analysis The peripheral lymphocytes were harvested by Ficoll Isopaque (Lympholyte Rat; Cedar Lane, Ontario, Canada) and cultured with 5 g/mL ConA (64H7090 Sigma, St Louis, Mo). After incubation for 48 hours, these cells were stained with FITC-conjugated mouse anti-rat CD4 antibody (clone W3/25; Serotec, Oxford) and biotinconjugated anti-rat CD25 antibody, and thereafter with streptavidin-perCP and propidium iodide. Then, the labeled cells were analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, Calif).
RESULTS AND DISCUSSION
MATERIALS AND METHODS Animals Adult male DA (RT1a) and Lewis (RT1l) rats were used as donors and recipients, respectively. The donor hearts were transplanted into a cervical location in the recipient using the cuff technique. Animals were divided into the following groups: group 1 (n ⫽ 6), recipients were administered 1 ⫻ 109 pfu of control vector, AxCALacZ (AdLacZ),4 via the tail vein immediately after transplantation; group 2 (n ⫽ 6), recipients orally administered FTY720 at a dose of 5 mg/kg 1 day before and the day of transplantation (day ⫺1 and 0); group 3 (n ⫽ 10), those administered 1 ⫻ 109 pfu of adenoviral vector containing CTLA4Ig gene, AxCAhCTLA4Ig (AdCTLA4Ig); group 4 (n ⫽ 10), recipients with combination therapy of above-mentioned administration schedules of AdCTLA4Ig and FTY720.
Histologic Studies The cardiac grafts were removed 5 days after transplantation for light microscopic study and immunochemistrical staining for CD2 (Mixture of MRC OX-54 and 55, Serotec, Oxford, England) in combination with CD25 (IL-2R alpha chain) antibody (clone
The recipients in group 1 rejected the grafts 6 to 7 days after grafting. On the other hand, the median survival time in group 2 was 9 days and that in group 3 was 24 days. The graft survival in group 4 was markedly prolonged to a median of 42.5 days (Table 1). No recipient death associated with adverse reaction was observed. Microscopic findings showed a marked infiltration of mononuclear cells in group 1, whereas little cell infiltration was observed in groups 3 and 4. The CD2 and CD25 From the Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center (Y.K., X.-K.L., N.F., S.E., A.T., H.A., S.S.), Tokyo, Department of Surgery II, Nagoya University, School of Medicine (S.H.), Nagoya, and First Department of Surgery, Hamamatsu University School of Medicine (K.S., T.K.), Hamamatsu, Japan. Address reprint requests to Xiao-Kang Li, MD, Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo, 154-8509 Japan.
Table 1. Effect of Combination Therapy with AxCAhCTLA4Ig and FTY720 on Rat Heart Transplantation Group
1. 2. 3. 4.
AdLacZ (control) FTY720 AdCTLA4Ig AdCTLA4Ig ⫹ FTY720
n
Survival (d)
P Value*
6 6 10 10
6 ⫻ 5, 7 8 ⫻ 2, 9 ⫻ 2, 13, 14 20, 21, 22 ⫻ 2, 24 ⫻ 2, 27 ⫻ 2, 28, 40 21, 32, 33, 40, 42, 43, 54, 56, ⬎100 ⫻ 2
⬍.05 ⬍.001 ⬍.001
*Welch’s unpaired t test was used for determining significant differences in survival rate between control group and experimental groups. P values are expressed vs group 1.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/99/$–see front matter PII S0041-1345(99)00567-9
Transplantation Proceedings, 31, 2787–2788 (1999)
2787
2788
double-positive cells were prominent in group 1, while very few were prominent in group 4. CD2 was expressed on both resting and activated T cells, while CD25 was only expressed on activated T cells. There were a number of TUNEL-positive cells (apoptotic cells) in the mononuclear cells around the blood vessels in groups 1 and 2. The apoptotic cells were very few in groups 3 and 4. The flow cytometric analysis in the ConAstimulated peripheral lymphocytes showed that CD25 expression was markedly deppressed in group 4 in comparison to the other groups. We considered the inhibition of T-cell activation by AdCTLA4Ig combined with the induction of lymphocyte apoptosis by FTY720 could markedly prolong the graft survival. Therefore, this study demonstrated that AdCTLA4Ig-gene transfection and perioperative adminis-
KITA, LI, FUNESHIMA ET AL
tration of FTY720 resulted in a combined immunosuppressive effect in rat cardiac allografting. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr H. Kimura and Dr T. Okuyama for their critical comments and useful suggestions. We also thank Dr I. Saito for providing AxCALacZ and Dr H. Hamada for providing the AxCAhCTLA4Ig adenoviral vector.
REFERENCES 1. Wu Y, Guo Y, Liu Y: J Exp Med 178:1789, 1993 2. Suzuki S, Enosawa S, Kakefuda T, et al: Transplantation 61:200, 1996 3. Suzuki S, Li X-K, Enosawa S, et al: Immunology 89:518, 1996 4. Kanegae Y, Lee G, Sato Y: Nucleic Acids Res 23:3816, 1995
C
TLA4Ig is a recombinant fusion protein that contains an extracellular domain of human CTLA4 and an Fc portion of human IgG1 heavy chain and blocks the B7/ CD28-mediated costimulatory signal, resulting in inhibition of T-lymphocyte activation.1 FTY720 is a synthetic drug produced by chemical modification of ISP-1 purified from culture filtrates of Isaria sinclairii. A single administration of FTY720 resulted in a marked reduction of peripheral lymphocytes.2,3 This study was conducted to define the combination effect of CTLA4Ig-gene transfection and perioperative administration of FTY720 in rat heart allografting.
OX-39; PharMingen, San Diego, Calif). We also stained apoptotic cells by the DNA nick-end labeling (TUNEL) method.
Flow Cytometric Analysis The peripheral lymphocytes were harvested by Ficoll Isopaque (Lympholyte Rat; Cedar Lane, Ontario, Canada) and cultured with 5 g/mL ConA (64H7090 Sigma, St Louis, Mo). After incubation for 48 hours, these cells were stained with FITC-conjugated mouse anti-rat CD4 antibody (clone W3/25; Serotec, Oxford) and biotinconjugated anti-rat CD25 antibody, and thereafter with streptavidin-perCP and propidium iodide. Then, the labeled cells were analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, Calif).
RESULTS AND DISCUSSION
MATERIALS AND METHODS Animals Adult male DA (RT1a) and Lewis (RT1l) rats were used as donors and recipients, respectively. The donor hearts were transplanted into a cervical location in the recipient using the cuff technique. Animals were divided into the following groups: group 1 (n ⫽ 6), recipients were administered 1 ⫻ 109 pfu of control vector, AxCALacZ (AdLacZ),4 via the tail vein immediately after transplantation; group 2 (n ⫽ 6), recipients orally administered FTY720 at a dose of 5 mg/kg 1 day before and the day of transplantation (day ⫺1 and 0); group 3 (n ⫽ 10), those administered 1 ⫻ 109 pfu of adenoviral vector containing CTLA4Ig gene, AxCAhCTLA4Ig (AdCTLA4Ig); group 4 (n ⫽ 10), recipients with combination therapy of above-mentioned administration schedules of AdCTLA4Ig and FTY720.
Histologic Studies The cardiac grafts were removed 5 days after transplantation for light microscopic study and immunochemistrical staining for CD2 (Mixture of MRC OX-54 and 55, Serotec, Oxford, England) in combination with CD25 (IL-2R alpha chain) antibody (clone
The recipients in group 1 rejected the grafts 6 to 7 days after grafting. On the other hand, the median survival time in group 2 was 9 days and that in group 3 was 24 days. The graft survival in group 4 was markedly prolonged to a median of 42.5 days (Table 1). No recipient death associated with adverse reaction was observed. Microscopic findings showed a marked infiltration of mononuclear cells in group 1, whereas little cell infiltration was observed in groups 3 and 4. The CD2 and CD25 From the Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center (Y.K., X.-K.L., N.F., S.E., A.T., H.A., S.S.), Tokyo, Department of Surgery II, Nagoya University, School of Medicine (S.H.), Nagoya, and First Department of Surgery, Hamamatsu University School of Medicine (K.S., T.K.), Hamamatsu, Japan. Address reprint requests to Xiao-Kang Li, MD, Department of Experimental Surgery and Bioengineering, National Children’s Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo, 154-8509 Japan.
Table 1. Effect of Combination Therapy with AxCAhCTLA4Ig and FTY720 on Rat Heart Transplantation Group
1. 2. 3. 4.
AdLacZ (control) FTY720 AdCTLA4Ig AdCTLA4Ig ⫹ FTY720
n
Survival (d)
P Value*
6 6 10 10
6 ⫻ 5, 7 8 ⫻ 2, 9 ⫻ 2, 13, 14 20, 21, 22 ⫻ 2, 24 ⫻ 2, 27 ⫻ 2, 28, 40 21, 32, 33, 40, 42, 43, 54, 56, ⬎100 ⫻ 2
⬍.05 ⬍.001 ⬍.001
*Welch’s unpaired t test was used for determining significant differences in survival rate between control group and experimental groups. P values are expressed vs group 1.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/99/$–see front matter PII S0041-1345(99)00567-9
Transplantation Proceedings, 31, 2787–2788 (1999)
2787
2788
double-positive cells were prominent in group 1, while very few were prominent in group 4. CD2 was expressed on both resting and activated T cells, while CD25 was only expressed on activated T cells. There were a number of TUNEL-positive cells (apoptotic cells) in the mononuclear cells around the blood vessels in groups 1 and 2. The apoptotic cells were very few in groups 3 and 4. The flow cytometric analysis in the ConAstimulated peripheral lymphocytes showed that CD25 expression was markedly deppressed in group 4 in comparison to the other groups. We considered the inhibition of T-cell activation by AdCTLA4Ig combined with the induction of lymphocyte apoptosis by FTY720 could markedly prolong the graft survival. Therefore, this study demonstrated that AdCTLA4Ig-gene transfection and perioperative adminis-
KITA, LI, FUNESHIMA ET AL
tration of FTY720 resulted in a combined immunosuppressive effect in rat cardiac allografting. ACKNOWLEDGMENTS The authors gratefully acknowledge Dr H. Kimura and Dr T. Okuyama for their critical comments and useful suggestions. We also thank Dr I. Saito for providing AxCALacZ and Dr H. Hamada for providing the AxCAhCTLA4Ig adenoviral vector.
REFERENCES 1. Wu Y, Guo Y, Liu Y: J Exp Med 178:1789, 1993 2. Suzuki S, Enosawa S, Kakefuda T, et al: Transplantation 61:200, 1996 3. Suzuki S, Li X-K, Enosawa S, et al: Immunology 89:518, 1996 4. Kanegae Y, Lee G, Sato Y: Nucleic Acids Res 23:3816, 1995