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− mice: a role for CD4+ t cells
Rejection of Grafts Without Histocompatibility Antigen Disparity by TAP1ⴚ/ⴚ Mice: A Role for CD4ⴙ T Cells I. Marrero, L.A. Benvenutti, I. Noronha, J. Kalil, and V. Coelho ABSTRACT We have previously shown that TAP1⫺/⫺ mice reject heart and skin grafts lacking an H-2 disparity. TAP1⫺/⫺ mice, which are deficient for MHC-I molecules, probably have a T-cell repertoire with distinct reactivity to these molecules. We speculated that this rejection could be mediated by CD4⫹ T cells reactive to H-2b class I molecules, or to class I– derived peptides presented by self-APC. This hypothesis was tested in the present work. Presensitization of TAP1⫺/⫺ mice with H-2Kb peptides accelerated the rejection of C57BL/6 (H-2b) skin grafts (MST 13 days, P ⬍ .0057), indicating that these peptides were able to mobilize effector T cells that participate in rejection. In addition, CD4 T-cell depletion before transplantation induced a significant delay in rejection (P ⬍ .0011), showing that CD4 T cells have a major role in the rejection process, though other cells may also contribute. In conclusion, these results support our hypothesis that H-2b molecules may be targeted in graft rejection without an H-2 disparity. The low expression of MHC-I molecules on TAP1⫺/⫺ mice may determine the selection of a T-cell repertoire that is reactive to self-MHC-I molecules, a phenomenon that is probably beyond the control of peripheral regulatory mechanisms.
T
AP1⫺/⫺ mice show defective intracellular transport of peptides due to targeted deletion of the TAP1 gene.1 As a consequence, TAP1⫺/⫺ mice have reduced numbers of MHC class I molecules on the cell surface and low numbers of mature CD8⫹ T cells in the periphery.1,2 It has been reported in the literature that cells from TAP1⫺/⫺, 2m⫺/⫺, and TAP12m⫺/⫺ mice develop CTL activity against target cells expressing normal levels of H-2b class I molecules.3,4 We have previously shown that TAP1⫺/⫺ mice reject grafts from littermate (TAP1⫹/⫺) and C57BL/6 (B6) donors.5 The predominance of CD4⫹ T cells and barely CD8⫹ cells infiltrating the graft, together with no expansion of CD8⫹ T cells in the periphery, suggested that CD4⫹ T cells play an important role in this rejection model.5 We speculated that TAP1⫺/⫺ mice that developed and matured in an MHC class I– deficient thymic environment may have selected a repertoire of T cells with distinct reactivity against H-2b class I molecules or derived peptides. Therefore, in the inflammatory context of transplantation of grafts with a relatively high density of class I molecules, TAP1⫺/⫺ mice may develop an inflammatory reaction. The aim of the present study was to investigate the role of CD4⫹ T cells in the rejection of B6 grafts by TAP1⫺/⫺ mice and whether H-2Kb class I peptides would be target molecules in this model of rejection. © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 999 –1000 (2004)
MATERIALS AND METHODS Animals TAP1⫺/⫺ (H-2b) mice were kindly provided by Dr L.V. Kaer (Vanderbilt University School of Medicine, Nashville, Tenn, USA). All mice were kept in an autoclaved isolator and treated in accordance with institutional ethical guidelines.
Synthetic Peptides Five 15mer MHC class I H-2Kb peptides, residues 39 to 53, 54 to 68, 69 to 83, 84 to 98, and 103 to 117, were synthesized in our laboratory using published sequences for H-2Kb MHC class I molecule.6
From the Immunology Laboratory, and Department of Pathology, Heart Institute (InCor), and Renal Division, Sa˜o Paulo University School of Medicine, Institute for Investigation in Immunology, Millennium Institute, Sa˜o Paulo, Brazil. This work was supported by grants from FAPESP (98/10632-2 and 98/10631-6), Sa˜o Paulo, Brazil. Address reprint requests to Veroˆnica Coelho, Laborato´rio de Imunologia, Instituto do Corac¸a˜o (InCor), HC-FMUSP, Av Dr Ene´as de Carvalho Aguiar-44, 9° andar, Sa˜o Paulo, 05403-001, SP, Brazil. E-mail: [email protected] 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.03.049 999
1000
MARRERO, BENVENUTTI, NORONHA ET AL
Peptide Immunization and Skin Grafting Mice were subcutaneously injected with 100 g of a mixture of H-2Kb peptides (20 g/peptide) emulsified in Complete Freund’s adjuvant (CFA) and 10 days later, restimulated with the same dose of peptides. After 10 days, these mice were transplanted with B6 tail skin grafts. The controls included mice injected with CFA alone or an unrelated OVA peptide, as well as mice injected with H-2Kb peptides and later transplanted with syngeneic TAP1⫺/⫺ skins.
CD4⫹ T-Cell Depletion and Skin Grafting TAP1⫺/⫺ mice were injected intraperitoneally with three doses of 100 g of IgG2b rat anti-murine CD4 mAb (GK1.5, PharMingen, La Jolla, Calif, USA). After to these mice were then transplantation with B6 skin grafts. The efficacy of CD4 depletion was evaluated by FACS.
RESULTS
H-2Kb-immunized TAP1⫺/⫺ mice displayed accelerated B6 graft rejection (MST ⫽ 13 days), compared to 16 days for nonimmunized TAP1⫺/⫺ mice (P ⬍ .0057). The effect of the peptides was specific: immunization with adjuvant alone or an unrelated OVA peptide did not accelerate graft rejection. On the other hand, TAP1⫺/⫺ mice immunized with H-2Kb peptides did not reject syngeneic TAP1⫺/⫺ grafts. Depletion of CD4⫹ T cells resulted in a significantly prolonged graft survival (MST ⫽ 41 days, P ⬍ .0011), compared to untreated controls (MST ⫽ 16 days). CD4 depletion of graft recipients resulted in long-term survival in 40% of the grafts (Fig. 1). These mice maintained B6 grafts without macroscopic evidence of rejection for more than 100 days after transplantation. Long-surviving B6 grafts showed no expression of IFN-␥ compared with high expression of IFN-␥ in rejected grafts. DISCUSSION
Fig 1. Depletion of CD4⫹ T cells delayed the rejection of B6 skin grafts. For depletion of CD4⫹ T cells, TAP1⫺/⫺ mice were injected with three doses of 100 g of anti-CD4 mAb (GK1.5) intraperitoneally and engrafted 10 days later (F, MST 41 days, n ⫽ 5). Graft survival was significantly higher than in nontreated recipients (}, MST 16 days, n ⫽ 18) (P ⫽ .0011).
infiltrating cells producing IFN-␥ were negatively regulated, suggesting that this cytokine could be implicated in the inflammatory response to the graft. In summary, our results show the important role of H-2b MHC class I molecules as targets in B6 graft rejection by TAP1⫺/⫺ mice. Our data also indicate that CD4⫹ T cells have an important role in this rejection model, possibly through the recognition of H-2Kb peptides presented by self-APC. REFERENCES
b
This study showed that immunization with H-2K peptides of TAP1⫺/⫺ mice resulted in a significant acceleration of B6 graft rejection, indicating that these peptides were able to prime a population of effector T cells that participate in the rejection process. Furthermore, depletion of CD4⫹ T cells resulted in delayed skin graft rejection inducing prolonged graft survival in 40% of recipients. These results demonstrate an important role of CD4⫹ T cells as effector cells in this rejection model. The lack of IFN-␥ in long-term accepted B6 grafts of the CD4-depleted group indicate that
1. Van Kaer L, Ashton-Rickardt PG, Ploegh HL, et al: Cell 71:1205, 1992 2. Ashton-Rickardt PG, Van Kaer L, Schumacher TNM, et al.: Cell 73:1041, 1993 3. Zijlstra M, Auchincloss H, Loring JM, et al: J Exp Med 175:885, 1992 4. Ljunggren HG, Van Kaer L, Sabatine MS, et al: Int Immunol 7:975, 1995 5. Coelho V, Marrero I, Noronha I, et al: Transplant Proc 31:900, 1999 6. Reyes AA, Schold M, Itakura K, et al: Proc Natl Acad Sci U S A 79:3270, 1992
T
AP1⫺/⫺ mice show defective intracellular transport of peptides due to targeted deletion of the TAP1 gene.1 As a consequence, TAP1⫺/⫺ mice have reduced numbers of MHC class I molecules on the cell surface and low numbers of mature CD8⫹ T cells in the periphery.1,2 It has been reported in the literature that cells from TAP1⫺/⫺, 2m⫺/⫺, and TAP12m⫺/⫺ mice develop CTL activity against target cells expressing normal levels of H-2b class I molecules.3,4 We have previously shown that TAP1⫺/⫺ mice reject grafts from littermate (TAP1⫹/⫺) and C57BL/6 (B6) donors.5 The predominance of CD4⫹ T cells and barely CD8⫹ cells infiltrating the graft, together with no expansion of CD8⫹ T cells in the periphery, suggested that CD4⫹ T cells play an important role in this rejection model.5 We speculated that TAP1⫺/⫺ mice that developed and matured in an MHC class I– deficient thymic environment may have selected a repertoire of T cells with distinct reactivity against H-2b class I molecules or derived peptides. Therefore, in the inflammatory context of transplantation of grafts with a relatively high density of class I molecules, TAP1⫺/⫺ mice may develop an inflammatory reaction. The aim of the present study was to investigate the role of CD4⫹ T cells in the rejection of B6 grafts by TAP1⫺/⫺ mice and whether H-2Kb class I peptides would be target molecules in this model of rejection. © 2004 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 36, 999 –1000 (2004)
MATERIALS AND METHODS Animals TAP1⫺/⫺ (H-2b) mice were kindly provided by Dr L.V. Kaer (Vanderbilt University School of Medicine, Nashville, Tenn, USA). All mice were kept in an autoclaved isolator and treated in accordance with institutional ethical guidelines.
Synthetic Peptides Five 15mer MHC class I H-2Kb peptides, residues 39 to 53, 54 to 68, 69 to 83, 84 to 98, and 103 to 117, were synthesized in our laboratory using published sequences for H-2Kb MHC class I molecule.6
From the Immunology Laboratory, and Department of Pathology, Heart Institute (InCor), and Renal Division, Sa˜o Paulo University School of Medicine, Institute for Investigation in Immunology, Millennium Institute, Sa˜o Paulo, Brazil. This work was supported by grants from FAPESP (98/10632-2 and 98/10631-6), Sa˜o Paulo, Brazil. Address reprint requests to Veroˆnica Coelho, Laborato´rio de Imunologia, Instituto do Corac¸a˜o (InCor), HC-FMUSP, Av Dr Ene´as de Carvalho Aguiar-44, 9° andar, Sa˜o Paulo, 05403-001, SP, Brazil. E-mail: [email protected] 0041-1345/04/$–see front matter doi:10.1016/j.transproceed.2004.03.049 999
1000
MARRERO, BENVENUTTI, NORONHA ET AL
Peptide Immunization and Skin Grafting Mice were subcutaneously injected with 100 g of a mixture of H-2Kb peptides (20 g/peptide) emulsified in Complete Freund’s adjuvant (CFA) and 10 days later, restimulated with the same dose of peptides. After 10 days, these mice were transplanted with B6 tail skin grafts. The controls included mice injected with CFA alone or an unrelated OVA peptide, as well as mice injected with H-2Kb peptides and later transplanted with syngeneic TAP1⫺/⫺ skins.
CD4⫹ T-Cell Depletion and Skin Grafting TAP1⫺/⫺ mice were injected intraperitoneally with three doses of 100 g of IgG2b rat anti-murine CD4 mAb (GK1.5, PharMingen, La Jolla, Calif, USA). After to these mice were then transplantation with B6 skin grafts. The efficacy of CD4 depletion was evaluated by FACS.
RESULTS
H-2Kb-immunized TAP1⫺/⫺ mice displayed accelerated B6 graft rejection (MST ⫽ 13 days), compared to 16 days for nonimmunized TAP1⫺/⫺ mice (P ⬍ .0057). The effect of the peptides was specific: immunization with adjuvant alone or an unrelated OVA peptide did not accelerate graft rejection. On the other hand, TAP1⫺/⫺ mice immunized with H-2Kb peptides did not reject syngeneic TAP1⫺/⫺ grafts. Depletion of CD4⫹ T cells resulted in a significantly prolonged graft survival (MST ⫽ 41 days, P ⬍ .0011), compared to untreated controls (MST ⫽ 16 days). CD4 depletion of graft recipients resulted in long-term survival in 40% of the grafts (Fig. 1). These mice maintained B6 grafts without macroscopic evidence of rejection for more than 100 days after transplantation. Long-surviving B6 grafts showed no expression of IFN-␥ compared with high expression of IFN-␥ in rejected grafts. DISCUSSION
Fig 1. Depletion of CD4⫹ T cells delayed the rejection of B6 skin grafts. For depletion of CD4⫹ T cells, TAP1⫺/⫺ mice were injected with three doses of 100 g of anti-CD4 mAb (GK1.5) intraperitoneally and engrafted 10 days later (F, MST 41 days, n ⫽ 5). Graft survival was significantly higher than in nontreated recipients (}, MST 16 days, n ⫽ 18) (P ⫽ .0011).
infiltrating cells producing IFN-␥ were negatively regulated, suggesting that this cytokine could be implicated in the inflammatory response to the graft. In summary, our results show the important role of H-2b MHC class I molecules as targets in B6 graft rejection by TAP1⫺/⫺ mice. Our data also indicate that CD4⫹ T cells have an important role in this rejection model, possibly through the recognition of H-2Kb peptides presented by self-APC. REFERENCES
b
This study showed that immunization with H-2K peptides of TAP1⫺/⫺ mice resulted in a significant acceleration of B6 graft rejection, indicating that these peptides were able to prime a population of effector T cells that participate in the rejection process. Furthermore, depletion of CD4⫹ T cells resulted in delayed skin graft rejection inducing prolonged graft survival in 40% of recipients. These results demonstrate an important role of CD4⫹ T cells as effector cells in this rejection model. The lack of IFN-␥ in long-term accepted B6 grafts of the CD4-depleted group indicate that
1. Van Kaer L, Ashton-Rickardt PG, Ploegh HL, et al: Cell 71:1205, 1992 2. Ashton-Rickardt PG, Van Kaer L, Schumacher TNM, et al.: Cell 73:1041, 1993 3. Zijlstra M, Auchincloss H, Loring JM, et al: J Exp Med 175:885, 1992 4. Ljunggren HG, Van Kaer L, Sabatine MS, et al: Int Immunol 7:975, 1995 5. Coelho V, Marrero I, Noronha I, et al: Transplant Proc 31:900, 1999 6. Reyes AA, Schold M, Itakura K, et al: Proc Natl Acad Sci U S A 79:3270, 1992