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Adoptive transfer of CD4 T-cell dependent allograft tolerance
ELSEVIER
Adoptive Transfer of CD4 T-Cell Dependent Allograft Tolerance M. Coulombe,
H. Yang, and R.G. Gill
P
RETREATMENT of C57BW6 (B6, H-2b) pancreatic islets in 95% oxygen culture leads to indefinite allograft acceptance (~100 d) in immunecompetent BALB/c (H-2d) recipients. Such pretreated grafts are composed of MHC Class I+II- parenchymal cells and are devoid of hematopoietic antigen presenting cells (APCs).’ The rejection of these APC-depleted grafts, induced by host immunization with donor-type spleen cells as a source of APCs, is CD8 T-cell mediated. Over time, a non-deletional form of donor-specific tolerance develops that can be adoptively transferred to immune-deficient C.B-17scid mice.* In this study, we determined the phenotype (CD4+ versus CD8+) of lymphocytes responsible for the tolerant state. Results show that tolerance resides in the CD4+ compartment and not in the CD8+ subset. MATERIALS AND METHODS Generation of Tolerant Animals C57BIJ6 (B6, H-2b) islets were depleted of APCs by a combination of cyclophosphamide-pretreatment of donors and a 7-day culture period in 95% OJS% CO,.* Streptozotocin-induced diabetic BALB/c mice received 400 cultured islets beneath the left kidney capsule. Graft function was assessed by frequent blood glucose monitoring. Rejection was indicated by a return to hyperglycemia. Animals that resisted graft rejection after challenge with donortype APCs 120 days after grafting were considered tolerant.’ Cell Fractionation
Spleen cells from tolerant or control mice were depleted of CD4+ T cells by incubation with anti-CD4 monoclonal antibody (10 CLg/mLGK1.5 ascites, rat IgG2,) followed by 10 &mL anti-rat Ig and rabbit complement. CD8+ T cells were depleted by treatment with anti-CD8 monoclonal antibody (supernatant from ADH(4)15 cells, IgM) and complement. Depletion of T cell subsets was confirmed by flow cytometry. Adoptive Transfer
of Tolerance
Streptozotocin-induced diabetic C.B-17&d (SCID, H-2“) mice were grafted with 450 untreated (immunogenic) donor-type (B6) or third party (CBA, H-2k) islets. Within 1 to 2 weeks of transplantation, 3 x 10’ unfractionated spleen cells from tolerant or control BALB/c mice were adoptively transferred IP to SCID recipients. In other experiments, 1.5 X 10’CD&depleted (CD4+) plus 1.5 X 10’ CD4-depleted (CD8+) spleen cells from tolerant and/or control mice were transferred to islet-grafted SCID mice. Graft survival and rejection were assessed as indicated above. After 60 days of graft function, removal of the islet-bearing kidney resulted in a 0041-1315/97/$17.00 PII SO041 -1345(96)00517-9
return of hyperglycemia, dependent.
RESULTS
AND
indicating that euglycemia was graft-
DISCUSSION
The tolerant state from animals bearing APC-depleted islet allografts could be adoptively transferred to SCID mice in a donor-specific manner. Spleen cells from tolerant animals failed to reject most donor-type B6 grafts while rejecting third-party CBA grafts (2/12 versus 7/8 respectively, P = .004). SCID recipients reconstituted with control BALB/c spleen cells rejected both B6 and CBA islet grafts (9/11 versus 7/7 respectively, P = NS). We then used this adoptive transfer model to determine the T-cell phenotype responsible for the tolerant state. Spleen cells from tolerant or control animals were fractionated into populations containing CD4+ or CD8+ T cells. Transferring various combinations of these lymphoid subpopulations to SCID recipients indicated the following: (1) control CD4’ mixed with control CD8+ T cells rejected both donor-type and third party grafts (7/7 versus 5/5 respectively, P = NS); (2) tolerant CD4+ plus tolerant CD8+ cells accepted the majority of donor-type (B6) grafts while rejecting thirdparty CBA grafts (2/7 versus 5/5 respectively, P = .03); (3) control CD4+ mixed with tolerant CD8+ spleen cells collaborated to reject both donor-type and third-party CBA grafts (818 versus 6/7 respectively, P = NS); and (4) tolerant CD4+ and control CD8+ spleen cells failed to collaborate to reject the majority of donor-type B6 grafts but were able to reject third-party CBA grafts (3/9 versus 7/8 respectively, P = .05). These results indicate that the tolerant phenotype maps to the CD4+ and not the CD8+ T cell subset, despite the fact that the original cultured allograft does not express Class II MHC antigens. Thus, rather than impairing the Class I-restricted CD8+ T cell subset, the tolerant state induced by the MHC Class I+II- islet graft paradoxically
From the Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO. Supported in part by National Institutes of Health grant ROl DK33470. Address reprint requests to Dr Ronald G. Gill, Barbara Davis Center for Childhood Diabetes, Univ of Colorado Health Science Center, 4200 E 9th Ave, Box B140, Denver, CO 80262.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1166
TransplantationProceedings, 29, 1166-l 167 (1997)
ADOPTIVE TRANSFER
leads to tolerance in the CD4+ T cell compartment. We hypothesize that this process occurs via the indirect pathway of antigen presentation in which CD4’ T cells see processed graft antigen in association with host Class II MHC leading to a regulatory response.3S4 Such regulatory T cells may interfere with the graft-specific CDs+ T cell response in vivo.
1167
REFERENCES
1. Parr E, Bowen K, Lafferty K: Transplantation 2. Coulombe M, Gill RG: Transplantation
30:135, 1980
57:1201, 1994
3. Shoskes DA, Wood KJ: Immunol Today 15:32, 1994 4. Gill RG, Coulombe M, Lafferty KJ: Immunol Rev 149:75, 1996
Adoptive Transfer of CD4 T-Cell Dependent Allograft Tolerance M. Coulombe,
H. Yang, and R.G. Gill
P
RETREATMENT of C57BW6 (B6, H-2b) pancreatic islets in 95% oxygen culture leads to indefinite allograft acceptance (~100 d) in immunecompetent BALB/c (H-2d) recipients. Such pretreated grafts are composed of MHC Class I+II- parenchymal cells and are devoid of hematopoietic antigen presenting cells (APCs).’ The rejection of these APC-depleted grafts, induced by host immunization with donor-type spleen cells as a source of APCs, is CD8 T-cell mediated. Over time, a non-deletional form of donor-specific tolerance develops that can be adoptively transferred to immune-deficient C.B-17scid mice.* In this study, we determined the phenotype (CD4+ versus CD8+) of lymphocytes responsible for the tolerant state. Results show that tolerance resides in the CD4+ compartment and not in the CD8+ subset. MATERIALS AND METHODS Generation of Tolerant Animals C57BIJ6 (B6, H-2b) islets were depleted of APCs by a combination of cyclophosphamide-pretreatment of donors and a 7-day culture period in 95% OJS% CO,.* Streptozotocin-induced diabetic BALB/c mice received 400 cultured islets beneath the left kidney capsule. Graft function was assessed by frequent blood glucose monitoring. Rejection was indicated by a return to hyperglycemia. Animals that resisted graft rejection after challenge with donortype APCs 120 days after grafting were considered tolerant.’ Cell Fractionation
Spleen cells from tolerant or control mice were depleted of CD4+ T cells by incubation with anti-CD4 monoclonal antibody (10 CLg/mLGK1.5 ascites, rat IgG2,) followed by 10 &mL anti-rat Ig and rabbit complement. CD8+ T cells were depleted by treatment with anti-CD8 monoclonal antibody (supernatant from ADH(4)15 cells, IgM) and complement. Depletion of T cell subsets was confirmed by flow cytometry. Adoptive Transfer
of Tolerance
Streptozotocin-induced diabetic C.B-17&d (SCID, H-2“) mice were grafted with 450 untreated (immunogenic) donor-type (B6) or third party (CBA, H-2k) islets. Within 1 to 2 weeks of transplantation, 3 x 10’ unfractionated spleen cells from tolerant or control BALB/c mice were adoptively transferred IP to SCID recipients. In other experiments, 1.5 X 10’CD&depleted (CD4+) plus 1.5 X 10’ CD4-depleted (CD8+) spleen cells from tolerant and/or control mice were transferred to islet-grafted SCID mice. Graft survival and rejection were assessed as indicated above. After 60 days of graft function, removal of the islet-bearing kidney resulted in a 0041-1315/97/$17.00 PII SO041 -1345(96)00517-9
return of hyperglycemia, dependent.
RESULTS
AND
indicating that euglycemia was graft-
DISCUSSION
The tolerant state from animals bearing APC-depleted islet allografts could be adoptively transferred to SCID mice in a donor-specific manner. Spleen cells from tolerant animals failed to reject most donor-type B6 grafts while rejecting third-party CBA grafts (2/12 versus 7/8 respectively, P = .004). SCID recipients reconstituted with control BALB/c spleen cells rejected both B6 and CBA islet grafts (9/11 versus 7/7 respectively, P = NS). We then used this adoptive transfer model to determine the T-cell phenotype responsible for the tolerant state. Spleen cells from tolerant or control animals were fractionated into populations containing CD4+ or CD8+ T cells. Transferring various combinations of these lymphoid subpopulations to SCID recipients indicated the following: (1) control CD4’ mixed with control CD8+ T cells rejected both donor-type and third party grafts (7/7 versus 5/5 respectively, P = NS); (2) tolerant CD4+ plus tolerant CD8+ cells accepted the majority of donor-type (B6) grafts while rejecting thirdparty CBA grafts (2/7 versus 5/5 respectively, P = .03); (3) control CD4+ mixed with tolerant CD8+ spleen cells collaborated to reject both donor-type and third-party CBA grafts (818 versus 6/7 respectively, P = NS); and (4) tolerant CD4+ and control CD8+ spleen cells failed to collaborate to reject the majority of donor-type B6 grafts but were able to reject third-party CBA grafts (3/9 versus 7/8 respectively, P = .05). These results indicate that the tolerant phenotype maps to the CD4+ and not the CD8+ T cell subset, despite the fact that the original cultured allograft does not express Class II MHC antigens. Thus, rather than impairing the Class I-restricted CD8+ T cell subset, the tolerant state induced by the MHC Class I+II- islet graft paradoxically
From the Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, CO. Supported in part by National Institutes of Health grant ROl DK33470. Address reprint requests to Dr Ronald G. Gill, Barbara Davis Center for Childhood Diabetes, Univ of Colorado Health Science Center, 4200 E 9th Ave, Box B140, Denver, CO 80262.
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1166
TransplantationProceedings, 29, 1166-l 167 (1997)
ADOPTIVE TRANSFER
leads to tolerance in the CD4+ T cell compartment. We hypothesize that this process occurs via the indirect pathway of antigen presentation in which CD4’ T cells see processed graft antigen in association with host Class II MHC leading to a regulatory response.3S4 Such regulatory T cells may interfere with the graft-specific CDs+ T cell response in vivo.
1167
REFERENCES
1. Parr E, Bowen K, Lafferty K: Transplantation 2. Coulombe M, Gill RG: Transplantation
30:135, 1980
57:1201, 1994
3. Shoskes DA, Wood KJ: Immunol Today 15:32, 1994 4. Gill RG, Coulombe M, Lafferty KJ: Immunol Rev 149:75, 1996