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Intrathymic inoculation of donor B cells prolongs cardiac allograft survival by non-deletional mechanisms
Intrathymic Inoculation of Donor B Cells Prolongs Cardiac Allograft Survival by Non-Deletional Mechanisms M. Niimi, P.J. Morris, and K.J. Wood
I
NTRATHYMIC inoculation (IT) of donor alloantigen and concomitant immunosuppressive treatment results in the induction of immune unresponsiveness to alloantigen.1– 6 To examine the involvement of nondeletional mechanisms, fractionated splenocytes were injected into only one lobe of the thymus. METHODS Animals CBA (H2k) and C57BL/10 (H2b) mice were purchased from Harlan Ltd. (Bicester, UK) and bred at the Biomedical Service Unit, John Radcliffe Hospital. All mice used were between 8 and 12 weeks old in accordance with the Animals Act 1986.
Preparation of Cells Resting B (rB) cells and splenic dendritic cells (DCs) were purified from splenocytes as described.7 A typical preparation of rB cells as analysed by flow cytometry was found to be 95.7% major histocompatibility class (MHC) II1, 1.2% CD801, 2.5% CD861, and 92.3% sIg1. A typical preparation of splenic DCs was 92.2% MHC II1, 68.8% CD801, 88.2% CD861, 68% N4181, and 9.4% sIg1. In functional assays, DCs but not rB cells were able to stimulate proliferation of allogeneic T cell and generate cytotoxic T cells.7 T cells were made from spleen using a nylon wool column as described7 and were sorted negatively by FACS Vantage with turbo sort option (Becton Dickinson, Mountain View, Calif). Typical preparations as analysed by FACS was found to be .90% CD31, ,0.5% IA1, and ,1% Ig1.
Recipient Treatment Hearts from C57BL/10 (H2b) mice were transplanted into CBA (H2k) recipients.8 Mice were pretreated with 50 mg depleting anti-CD4 monoclonal antibody (mAb), YTA 3.1,9 27 and 28 days before grafting. With the second dose of mAb, 1 3 106 donor unfractionated or fractionated leukocytes [ie, rBs, DCs, and T cells] were injected into a single lobe or both lobes of thymus.10
RESULTS
When rBs, DCs, or T cells were delivered into a single lobe of the thymus with anti-CD4 mAb, only rBs induced indefinite prolongation of graft survival in all recipients (median survival time [MST] . 100 days); unfractionated splenocytes, DCs, and T cells were less effective (MST 5 62, 64, and 21 days, respectively) (P , 0.01). In contrast, IT of 0041-1345/99/$–see front matter PII S0041-1345(98)01454-7
any of the fractionated populations (rBs, DCs, and T cells) induced indefinite prolongation of cardiac allograft survival in all recipients when delivered into both thymic lobes with anti-CD4 mAb. Untreated mice or controls pretreated with anti-CD4 mAb alone rejected their grafts acutely (MST 5 7.5 and 12 days, respectively). We confirmed that cells injected into one lobe of the thymus did not migrate to the uninjected lobe by FACS analysis. Intravenous injection of 1 3 106 rBs with anti-CD4 mAb did not induce specific unresponsiveness (MST 5 30.5 days), suggesting that these data could not be explained by peripheral effects alone. Moreover, mice thymectomized in one thymic lobe were able to reject grafts even when treated with anti-CD4 mAb (MST 5 28 days). Therefore, one thymic lobe was sufficient to induce graft rejection. Taken together, these data suggest that regulatory mechanisms generated by intrathymic injection of donor rB cells suppressed the rejection response elicited from the uninjected lobe. CONCLUSION
IT of rBs induced prolongation of fully allogeneic cardiac grafts by a nondeletional mechanism. REFERENCES 1. Vojtiskova M, et al: Transplantation 6:13, 1968 2. Staples P, et al: J Exp Med 124:127, 1966 3. Posselt AM, et al: Science 249:1293, 1990 4. Goss JA, et al: Ann Surg 217:492, 1993 5. Oluwole SF, et al: Transplantation 55:1396, 1993 6. Knechtle SJ, et al: Transplantation 57:990, 1994 7. Niimi M, et al: Transplantation 64:1330, 1997 8. Corry RJ, et al: Transplantation 16:343, 1973 9. Cobbold SP, et al: Nature 312:548, 1984 10. Jones ND, et al: Eur J Immunol 27:1591, 1997 From the Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, England. This work was supported by The Wellcome Trust, Medical Research Council UK and the British Heart Foundation. MN is the recipient of a Teikyo scholarship. Address reprint requests to Kathryn J. Wood, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
94
Transplantation Proceedings, 31, 94 (1999)
I
NTRATHYMIC inoculation (IT) of donor alloantigen and concomitant immunosuppressive treatment results in the induction of immune unresponsiveness to alloantigen.1– 6 To examine the involvement of nondeletional mechanisms, fractionated splenocytes were injected into only one lobe of the thymus. METHODS Animals CBA (H2k) and C57BL/10 (H2b) mice were purchased from Harlan Ltd. (Bicester, UK) and bred at the Biomedical Service Unit, John Radcliffe Hospital. All mice used were between 8 and 12 weeks old in accordance with the Animals Act 1986.
Preparation of Cells Resting B (rB) cells and splenic dendritic cells (DCs) were purified from splenocytes as described.7 A typical preparation of rB cells as analysed by flow cytometry was found to be 95.7% major histocompatibility class (MHC) II1, 1.2% CD801, 2.5% CD861, and 92.3% sIg1. A typical preparation of splenic DCs was 92.2% MHC II1, 68.8% CD801, 88.2% CD861, 68% N4181, and 9.4% sIg1. In functional assays, DCs but not rB cells were able to stimulate proliferation of allogeneic T cell and generate cytotoxic T cells.7 T cells were made from spleen using a nylon wool column as described7 and were sorted negatively by FACS Vantage with turbo sort option (Becton Dickinson, Mountain View, Calif). Typical preparations as analysed by FACS was found to be .90% CD31, ,0.5% IA1, and ,1% Ig1.
Recipient Treatment Hearts from C57BL/10 (H2b) mice were transplanted into CBA (H2k) recipients.8 Mice were pretreated with 50 mg depleting anti-CD4 monoclonal antibody (mAb), YTA 3.1,9 27 and 28 days before grafting. With the second dose of mAb, 1 3 106 donor unfractionated or fractionated leukocytes [ie, rBs, DCs, and T cells] were injected into a single lobe or both lobes of thymus.10
RESULTS
When rBs, DCs, or T cells were delivered into a single lobe of the thymus with anti-CD4 mAb, only rBs induced indefinite prolongation of graft survival in all recipients (median survival time [MST] . 100 days); unfractionated splenocytes, DCs, and T cells were less effective (MST 5 62, 64, and 21 days, respectively) (P , 0.01). In contrast, IT of 0041-1345/99/$–see front matter PII S0041-1345(98)01454-7
any of the fractionated populations (rBs, DCs, and T cells) induced indefinite prolongation of cardiac allograft survival in all recipients when delivered into both thymic lobes with anti-CD4 mAb. Untreated mice or controls pretreated with anti-CD4 mAb alone rejected their grafts acutely (MST 5 7.5 and 12 days, respectively). We confirmed that cells injected into one lobe of the thymus did not migrate to the uninjected lobe by FACS analysis. Intravenous injection of 1 3 106 rBs with anti-CD4 mAb did not induce specific unresponsiveness (MST 5 30.5 days), suggesting that these data could not be explained by peripheral effects alone. Moreover, mice thymectomized in one thymic lobe were able to reject grafts even when treated with anti-CD4 mAb (MST 5 28 days). Therefore, one thymic lobe was sufficient to induce graft rejection. Taken together, these data suggest that regulatory mechanisms generated by intrathymic injection of donor rB cells suppressed the rejection response elicited from the uninjected lobe. CONCLUSION
IT of rBs induced prolongation of fully allogeneic cardiac grafts by a nondeletional mechanism. REFERENCES 1. Vojtiskova M, et al: Transplantation 6:13, 1968 2. Staples P, et al: J Exp Med 124:127, 1966 3. Posselt AM, et al: Science 249:1293, 1990 4. Goss JA, et al: Ann Surg 217:492, 1993 5. Oluwole SF, et al: Transplantation 55:1396, 1993 6. Knechtle SJ, et al: Transplantation 57:990, 1994 7. Niimi M, et al: Transplantation 64:1330, 1997 8. Corry RJ, et al: Transplantation 16:343, 1973 9. Cobbold SP, et al: Nature 312:548, 1984 10. Jones ND, et al: Eur J Immunol 27:1591, 1997 From the Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, England. This work was supported by The Wellcome Trust, Medical Research Council UK and the British Heart Foundation. MN is the recipient of a Teikyo scholarship. Address reprint requests to Kathryn J. Wood, Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
94
Transplantation Proceedings, 31, 94 (1999)