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Costimulatory signal blockade by anti-CD2 monoclonal antibody in combination with 15-deoxyspergualin prolongs concordant xenograft survival
Costimulatory Signal Blockade by Anti-CD2 Monoclonal Antibody in Combination With 15-Deoxyspergualin Prolongs Concordant Xenograft Survival M. Haga, H. Hirahara, M. Tsuchida, T. Watanabe, M. Takekubo, and J. Hayashi
A
NTIBODY treatment is now focused either on induction therapy or on rescue therapy for acute allogeneic rejection. Many investigators have shown that the use of monoclonal antibodies (mAb) can help achieve immunologic tolerance in allo- and xenotransplantation models.1–7 We have reported that short-term administration of antiCD2 mAb before or after transplantation prolongs graft survival in an allotransplantation model.1 However, fewer data have been reported on antibody treatment in xenotransplantation. Here we attempt to clarify the effects of costimulatory signal blockade by anti-rat CD2 mAb on a mouse-to-rat cardiac xenotransplantation model.
Antibody Preparation Hybridoma cells (OX34) that produce monoclonal antibody to rat CD2 were grown in culture and injected into pristane-primed BALB/c mice to produce ascites. MAb was then purified twice from the ascites by ammonium sulfate precipitation.
15-Deoxyspergualin The drug was supplied as a pure powder (Nippon Kayaku Co Ltd, Tokyo) and suspended in normal saline before use. To fill the osmotic pumps, 100 mg of the drug was formulated in normal saline according to body weight and administration term.
Xenogeneic Mixed Lymphocyte Reaction (MLR)
MATERIALS AND METHODS Animals and Experimental Design
T-cell– enriched splenocytes of rats were used as responders, irradiated splenocytes of BALB/c mice or BN rats as stimulators. Plated lymphocytes in microwell cultures were incubated with or
Heterotopic heart transplantations from BALB/c mice to Lewis rats were carried out on day 0. Rats were divided into four treatment groups: no treatment, anti-CD2 mAb, 15-deoxyspergualin (DSG) and combination treatment groups. Anti-CD2 mAb was given at 7 mg/kg/d once a day on days 0, 1, 3, and 5. DSG was administered intraperitoneally at 5 mg/kg/d using osmotic pumps (Alzea Corporation, Palo Alto, Calif) from day 0 to day 28.
From the Department of Thoracic and Cardiovascular Surgery, Niigata University School of Medicine, Niigata, Japan. Address reprint requests to Manabu Haga, MD, Department of Thoracic and Cardiovascular Surgery, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata City, 951-8510 Japan. E-mail: [email protected].
Fig 1. Effects of monoclonal antibody OX34 on allogeneic and xenogeneic MLR. Naive LEW spleen T cells (2 ⫻ 105) were cocultured with 2 ⫻ 105 irradiated LEW (syngeneic), BN (allogeneic), or BALB/c (xenogeneic) stimulator cells in flatbottom microwell plates for 4 days in the absence or presence of OX34 (1 L/mL). Data represent mean ⫾ SD of triplicate determinations.
0041-1345/00/$–see front matter PII S0041-1345(00)01086-1
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1006
Transplantation Proceedings, 32, 1006–1008 (2000)
COSTIMULATORY SIGNAL BLOCKADE
1007
Table 1. Effect of Combination Therapy of OX34 With DSG on Prolongation of Xenograft Survival Group
Treatment
n
Survival Time (d)
Mean Survival Time (d ⫾ SD)
p (vs group 1)
1 2 3 4
Non-treatment OX34* DSG† OX34 ⫹ DSG
12 10 10 10
3 (x9), 4 (x3) 3 (x8), 4, 4 5, 5, 6, 6, 6, 7, 7, 8, 8, 9 36, 39, 40, 48, 52 (x3), 55, 57
3.3 ⫾ 0.5 3.2 ⫾ 0.4 6.7 ⫾ 1.3 47.9 ⫾ 7.6
.8432 .0001 .0001
*Anti-CD2 mAb (OX34) was given at 7 mg/kg/d once a day on days 0, 1, 3, and 5. † DSG was administered intraperitoneally at the dose of 5 mg/kg/d by continuous infusion using osmotic pumps for 28 days just after xenotransplantation.
without anti-CD2 mAb in a 5% CO2 incubator at 37°C for 3 days, and cocultured with 0.5 Ci of 3[H] Thymidine (Amersham) for the 18 hours. The cells were harvested on glass-fiber filters and label uptake was determined using standard liquid scintillation techniques. Anti-mouse antibody titers were measured by flow cytometry using mouse thymocytes as targets. Briefly, thymocytes (1 ⫻ 106 cells) were incubated with 10 L of recipient serum in tenfold dilutions for 30 minutes on ice, and washed twice. The cells were incubated with FITC-conjugated anti-rat IgM or FITC-conjugated anti-rat IgG. After washing twice, the cells were analyzed by flow
cytometry. Antibody titers are presented as peak channel fluorescence intensity.
RESULTS Xenogeneic MLR
To determine whether anti-CD2 mAb (OX34) suppressed rat lymphocyte proliferation to xenoantigens, we assessed xenogeneic MLR using irradiated mouse splenocytes as stimulators. OX34 in mixed lymphocyte culture suppressed
Fig 2. Cardiac xenograft on day 50 in a recipient treated with OX34 and DSG (A) was indistinguishable from normal mouse heart (B). In contrast, rejected xenograft on day 52 in a recipient treated with OX34 and DSG (C) or xenograft undergoing rejection three days after transplantation without treatment (D) demonstrated interstitial hemorrhage, edema, and polymorphonuclear cell infiltration.
1008
HAGA, HIRAHARA, TSUCHIDA ET AL
Fig 3. Xenoreactive IgM and IgG titer. Rats without treatment (■) or treated with OX34 (F), DSG (Œ), and a combination of OX34 and DSG (}) received heterotopic cardiac transplantation. Sera were harvested every 1 to 5 days from the day of transplantation until rejection. Xenogeneic antibody titers are presented as peak channel fluorescence ⫾ SD (n ⫽ 4 –5).
rat lymphocyte proliferation to mouse splenocytes as well as allogeneic rat splenocytes (Fig 1). Graft Survival
Untreated control rats rejected mouse cardiac xenografts in 4 days. Anti-CD2 mAb alone could not prolong xenograft survival. DSG alone slightly prolonged xenograft survival to 6.7 ⫾ 1.3 days. Combination therapy of anti-CD2 mAb together with DSG dramatically improved xenograft survival to 44.7 ⫾ 7.6 days (Table 1). Histologic analysis revealed that the cause of rejection in all the groups was vascular rejection, which was characterized by hemorrhage, edema, and infiltration by polymorphonuclear cells (Fig 2). Antibody Titer
At the time of rejection, xenoreactive antibody titers, of both IgM and IgG isotypes, were increased in all treatment groups (Fig 3).
prolongation of xenograft survival. The second is suppression of T-cell– dependent and –independent antibody production by anti-CD2 mAb and DSG. Because single-agent anti-CD2 mAb or DSG could not completely inhibit xenoreactive antibody production, the synergism of both drugs is important. The mechanism of xenograft rejection in the combination therapy group is also an important problem. We speculate that partial blockade of T cells or abrogation of T-cell suppression may cause rejection, because IgG xenoreactive antibody titer, which is considered to be T cell– dependent, was increased, as well as the IgM titer, and because rat lymphocyte proliferation to mouse stimulators was not completely suppressed in MLR. On the other hand, rejection may be caused by effectors other than T cells, such as NK cells, macrophages, and T-independent antibody. These effectors must be ruled out in future studies. Through the analysis of these factors, refinement of this protocol may achieve permanent acceptance of xenografts and T-cell unresponsiveness toward xenografts in this model.
DISCUSSION
Costimulatory signal blockade by anti-CD2 mAb was effective in prolonging xenograft survival in combination with DSG. The mechanism by which such combination therapy prolonged xenograft survival in this study is not completely understood. Several mechanisms may be responsible for the prolongation. The first is inhibition of T-cell activation by anti-CD2 mAb. In allogeneic transplantation models, we suggested that anergy may be responsible for unresponsiveness.1 However, in xenografts, it is unclear how the antibody suppresses T-cell activation. Because the combination therapy critically decreased the number of T cells to less than 5% in the spleens on day 28 (data not shown), specific or nonspecific decreases in the T-cell population may cause
REFERENCES 1. Hirahara H, Tsuchida M, Watanabe T, et al: Transplantation 59:85, 1995 2. Shizuru JA, Seydel KB, Flavin TF, et al: Transplantation 50:366, 1990 3. Sayegh MH, Sablinski T, Tanaka K, et al: Transplantation 51:296, 1991 4. Chen Z, Cobbold S, Metcalfe S, et al: Eur J Immunol 22:805, 1992 5. Isobe M, Yagita H, Okumura K, et al: Science 255:1125, 1992 6. Nicolls MR, Awersa GG, Pearce NW, et al: Transplantation 55:459, 1993 7. Tsuchida M, Hirahara H, Matsumoto Y, et al: Transplantation 57:256, 1994
A
NTIBODY treatment is now focused either on induction therapy or on rescue therapy for acute allogeneic rejection. Many investigators have shown that the use of monoclonal antibodies (mAb) can help achieve immunologic tolerance in allo- and xenotransplantation models.1–7 We have reported that short-term administration of antiCD2 mAb before or after transplantation prolongs graft survival in an allotransplantation model.1 However, fewer data have been reported on antibody treatment in xenotransplantation. Here we attempt to clarify the effects of costimulatory signal blockade by anti-rat CD2 mAb on a mouse-to-rat cardiac xenotransplantation model.
Antibody Preparation Hybridoma cells (OX34) that produce monoclonal antibody to rat CD2 were grown in culture and injected into pristane-primed BALB/c mice to produce ascites. MAb was then purified twice from the ascites by ammonium sulfate precipitation.
15-Deoxyspergualin The drug was supplied as a pure powder (Nippon Kayaku Co Ltd, Tokyo) and suspended in normal saline before use. To fill the osmotic pumps, 100 mg of the drug was formulated in normal saline according to body weight and administration term.
Xenogeneic Mixed Lymphocyte Reaction (MLR)
MATERIALS AND METHODS Animals and Experimental Design
T-cell– enriched splenocytes of rats were used as responders, irradiated splenocytes of BALB/c mice or BN rats as stimulators. Plated lymphocytes in microwell cultures were incubated with or
Heterotopic heart transplantations from BALB/c mice to Lewis rats were carried out on day 0. Rats were divided into four treatment groups: no treatment, anti-CD2 mAb, 15-deoxyspergualin (DSG) and combination treatment groups. Anti-CD2 mAb was given at 7 mg/kg/d once a day on days 0, 1, 3, and 5. DSG was administered intraperitoneally at 5 mg/kg/d using osmotic pumps (Alzea Corporation, Palo Alto, Calif) from day 0 to day 28.
From the Department of Thoracic and Cardiovascular Surgery, Niigata University School of Medicine, Niigata, Japan. Address reprint requests to Manabu Haga, MD, Department of Thoracic and Cardiovascular Surgery, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata City, 951-8510 Japan. E-mail: [email protected].
Fig 1. Effects of monoclonal antibody OX34 on allogeneic and xenogeneic MLR. Naive LEW spleen T cells (2 ⫻ 105) were cocultured with 2 ⫻ 105 irradiated LEW (syngeneic), BN (allogeneic), or BALB/c (xenogeneic) stimulator cells in flatbottom microwell plates for 4 days in the absence or presence of OX34 (1 L/mL). Data represent mean ⫾ SD of triplicate determinations.
0041-1345/00/$–see front matter PII S0041-1345(00)01086-1
© 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1006
Transplantation Proceedings, 32, 1006–1008 (2000)
COSTIMULATORY SIGNAL BLOCKADE
1007
Table 1. Effect of Combination Therapy of OX34 With DSG on Prolongation of Xenograft Survival Group
Treatment
n
Survival Time (d)
Mean Survival Time (d ⫾ SD)
p (vs group 1)
1 2 3 4
Non-treatment OX34* DSG† OX34 ⫹ DSG
12 10 10 10
3 (x9), 4 (x3) 3 (x8), 4, 4 5, 5, 6, 6, 6, 7, 7, 8, 8, 9 36, 39, 40, 48, 52 (x3), 55, 57
3.3 ⫾ 0.5 3.2 ⫾ 0.4 6.7 ⫾ 1.3 47.9 ⫾ 7.6
.8432 .0001 .0001
*Anti-CD2 mAb (OX34) was given at 7 mg/kg/d once a day on days 0, 1, 3, and 5. † DSG was administered intraperitoneally at the dose of 5 mg/kg/d by continuous infusion using osmotic pumps for 28 days just after xenotransplantation.
without anti-CD2 mAb in a 5% CO2 incubator at 37°C for 3 days, and cocultured with 0.5 Ci of 3[H] Thymidine (Amersham) for the 18 hours. The cells were harvested on glass-fiber filters and label uptake was determined using standard liquid scintillation techniques. Anti-mouse antibody titers were measured by flow cytometry using mouse thymocytes as targets. Briefly, thymocytes (1 ⫻ 106 cells) were incubated with 10 L of recipient serum in tenfold dilutions for 30 minutes on ice, and washed twice. The cells were incubated with FITC-conjugated anti-rat IgM or FITC-conjugated anti-rat IgG. After washing twice, the cells were analyzed by flow
cytometry. Antibody titers are presented as peak channel fluorescence intensity.
RESULTS Xenogeneic MLR
To determine whether anti-CD2 mAb (OX34) suppressed rat lymphocyte proliferation to xenoantigens, we assessed xenogeneic MLR using irradiated mouse splenocytes as stimulators. OX34 in mixed lymphocyte culture suppressed
Fig 2. Cardiac xenograft on day 50 in a recipient treated with OX34 and DSG (A) was indistinguishable from normal mouse heart (B). In contrast, rejected xenograft on day 52 in a recipient treated with OX34 and DSG (C) or xenograft undergoing rejection three days after transplantation without treatment (D) demonstrated interstitial hemorrhage, edema, and polymorphonuclear cell infiltration.
1008
HAGA, HIRAHARA, TSUCHIDA ET AL
Fig 3. Xenoreactive IgM and IgG titer. Rats without treatment (■) or treated with OX34 (F), DSG (Œ), and a combination of OX34 and DSG (}) received heterotopic cardiac transplantation. Sera were harvested every 1 to 5 days from the day of transplantation until rejection. Xenogeneic antibody titers are presented as peak channel fluorescence ⫾ SD (n ⫽ 4 –5).
rat lymphocyte proliferation to mouse splenocytes as well as allogeneic rat splenocytes (Fig 1). Graft Survival
Untreated control rats rejected mouse cardiac xenografts in 4 days. Anti-CD2 mAb alone could not prolong xenograft survival. DSG alone slightly prolonged xenograft survival to 6.7 ⫾ 1.3 days. Combination therapy of anti-CD2 mAb together with DSG dramatically improved xenograft survival to 44.7 ⫾ 7.6 days (Table 1). Histologic analysis revealed that the cause of rejection in all the groups was vascular rejection, which was characterized by hemorrhage, edema, and infiltration by polymorphonuclear cells (Fig 2). Antibody Titer
At the time of rejection, xenoreactive antibody titers, of both IgM and IgG isotypes, were increased in all treatment groups (Fig 3).
prolongation of xenograft survival. The second is suppression of T-cell– dependent and –independent antibody production by anti-CD2 mAb and DSG. Because single-agent anti-CD2 mAb or DSG could not completely inhibit xenoreactive antibody production, the synergism of both drugs is important. The mechanism of xenograft rejection in the combination therapy group is also an important problem. We speculate that partial blockade of T cells or abrogation of T-cell suppression may cause rejection, because IgG xenoreactive antibody titer, which is considered to be T cell– dependent, was increased, as well as the IgM titer, and because rat lymphocyte proliferation to mouse stimulators was not completely suppressed in MLR. On the other hand, rejection may be caused by effectors other than T cells, such as NK cells, macrophages, and T-independent antibody. These effectors must be ruled out in future studies. Through the analysis of these factors, refinement of this protocol may achieve permanent acceptance of xenografts and T-cell unresponsiveness toward xenografts in this model.
DISCUSSION
Costimulatory signal blockade by anti-CD2 mAb was effective in prolonging xenograft survival in combination with DSG. The mechanism by which such combination therapy prolonged xenograft survival in this study is not completely understood. Several mechanisms may be responsible for the prolongation. The first is inhibition of T-cell activation by anti-CD2 mAb. In allogeneic transplantation models, we suggested that anergy may be responsible for unresponsiveness.1 However, in xenografts, it is unclear how the antibody suppresses T-cell activation. Because the combination therapy critically decreased the number of T cells to less than 5% in the spleens on day 28 (data not shown), specific or nonspecific decreases in the T-cell population may cause
REFERENCES 1. Hirahara H, Tsuchida M, Watanabe T, et al: Transplantation 59:85, 1995 2. Shizuru JA, Seydel KB, Flavin TF, et al: Transplantation 50:366, 1990 3. Sayegh MH, Sablinski T, Tanaka K, et al: Transplantation 51:296, 1991 4. Chen Z, Cobbold S, Metcalfe S, et al: Eur J Immunol 22:805, 1992 5. Isobe M, Yagita H, Okumura K, et al: Science 255:1125, 1992 6. Nicolls MR, Awersa GG, Pearce NW, et al: Transplantation 55:459, 1993 7. Tsuchida M, Hirahara H, Matsumoto Y, et al: Transplantation 57:256, 1994