- Email: [email protected]
The Role of Foxp3+ Regulatory T Cells in Liver Transplant Tolerance
The Role of Foxp3ⴙ Regulatory T Cells in Liver Transplant Tolerance W. Li, K. Carper, X.X. Zheng, C.S. Kuhr, J.D. Reyes, Y. Liang, D.L. Perkins, A.W. Thomson, and J.D. Perkins ABSTRACT The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4⫹CD25⫹ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4⫹CD25⫹ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3⫹ cells in the liver grafts and recipient spleens and increased transforming growth factor  expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4⫹CD25⫹ Treg using anti-CD25 monoclonal antibody (250 g/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-␥, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3⫹CD4⫹CD25⫹ Treg appear to underpin spontaneous acceptance of major histocompatability complex– mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.
P
REVIOUS FINDINGS suggest that the liver is a tolerogenic organ. Induction of hepatic tolerance is an active process, possibly mediated by regulatory T cells (Treg).1,2 CD4⫹CD25⫹ T cells, expressing Foxp3, have been proposed as an important regulator of both self- and transplantation tolerance.3 The role of these Treg in hepatic tolerance is unknown. Our objective was to characterize Foxp3⫹CD4⫹CD25⫹ Treg in liver allograft recipients and examine the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. MATERIALS AND METHODS Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice as described.1 Animals were sacrificed at various times from 5 to 100 days posttransplant for Treg characterization by four-color flow cytometry for Foxp3 and transforming growth
factor (TGF)  protein expression by immunohistochemistry and for tolerance transfer by adoptive transfer of spleen cells (SCs) from long-term liver allograft survivors to the C3H mice, which received B10 heart grafts. Rat anti-mouse CD25 monoclonal
From the Departments of Surgery (W.L., K.C., C.S.K., J.D.R., J.D.P.), and Urology (C.S.K.), Division of Transplantation, University of Washington Medical Center, Seattle, Washington, USA; Department of Medicine (X.X.Z.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; Departments of Medicine and Surgery (Y.L., D.L.P.), University of California San Diego, La Jolla, California, USA; and Department of Surgery (A.W.T.), Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. Address reprint requests to Wei Li, MD, PhD. Division of Transplantation, Department of Surgery, University of Washington Medical Center, Box 356174, 1959 NE Pacific Street, Seattle, Washington 98195. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.093
Transplantation Proceedings, 38, 3205–3206 (2006)
3205
3206 antibody (mAb; PC61; Chimerigen, Allston, Mass, USA; 250 g, intraperitoneally) was administered to liver recipients on days ⫺6, ⫺4, ⫺2 pre- or days 0, 1, 2, 3 posttransplant, respectively. Controls received rat immunoglobulin G (IgG) (250 g; Sigma, Milwaukee, Wis, USA) using an identical regimen. Hamster anti-mouse CTLA4 mAb (UC10-4F10-11) provided by Dr X.X. Zheng (Beth Israel Deaconess Medical Center, Boston, Mass, USA) was administered (250 g, intraperitoneally) on days 0 and 2 to the anti-CD25 mAb pretreated recipients. The following parameters were examined at day 5 posttransplantation: T-cell proliferative activity by MLR; CTL and natural killer (NK) activities by 4hr 51Cr release assay; cytokine interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-␥ production by Elispot; and apoptotic activity by TUNEL staining. Graft survival was analyzed using Kaplan-Maier and others were made using Students t test.
RESULTS
The percentage of CD4⫹CD25⫹ T cells within the CD3 cell population was increased in both liver grafts and recipient spleens posttransplantation compared to syngeneic control or naïve mice. Levels peaked within first 2 weeks. These CD4⫹CD25⫹ T cells expressed intracellular Foxp3 (⬎90%) and CTLA4 (⬎80%). The Foxp3 protein expression detected in frozen sections of liver grafts and host spleens was increased posttransplant from day 5 up to day 100, while TGF production was increased in the spleens posttransplant throughout the time courses compared to naïve mice. Adoptive transfer of the SCs from long-term liver allograft survivors to naïve C3H significantly prolonged B10 heart graft survival [median survival time (MST) ⬎30 days vs control 10 days, P ⬍ .001]. Depletion of recipient CD4⫹CD25⫹ Treg by administration of anti-CD25 mAb, either pre- (MST 14.9 days, n ⫽ 8, P ⬍ .01) or posttransplantation (MST 16.8 days, n ⫽ 6, P ⬍ .01), induced liver allograft acute rejection compared to IgG control. Moreover, CTLA4 blockade by anti-CTLA mAb prevented liver allograft spontaneous acceptance2 and exacerbated liver allograft rejection in anti-CD25 pretreated recipients (MST 7.8 days, n ⫽ 5, P ⬍ .05). In association with liver allograft acute rejection by depletion of CD4⫹CD25⫹ Treg, donor-specific proliferative responses of T cells and anti-donor CTL and NK activities of liver graft infiltrating cells and SCs from anti-CD25 mAbtreated recipients were significantly increased compared with those of cells from control mice. Depletion of Treg also decreased the number of IL-4-producing cells, pro-
LI, CARPER, ZHENG ET AL
moted expansion of IL-2-, IFN-␥-, and IL-10-producing cells, and reduced apoptosis of T cells in the liver grafts and recipient spleens at day 5 posttransplantation. DISCUSSION
Our results suggest that CD4⫹CD25⫹ Treg are expanded after liver transplantation and play a key role in inducing or maintaining “spontaneous” liver transplant tolerance. CTLA4 and TGF may be the key factors in Treg-mediated immune suppression.2– 4 Depletion of recipient CD4⫹CD25⫹ Treg by administration of anti-CD25 mAb significantly reduced the survival of liver allografts. This is also confirmed in major histocompatability complex–mismatched heart and skin transplant models.5 Moreover, anti-CD25 mAb administration plus CTLA4 blockade accelerated liver graft rejection compared to anti-CD25 treatment alone, suggesting that CTLA4 may be involved in CD4⫹CD25⫹ Treg functions.3,4 Our findings also provide evidence that CD4⫹CD25⫹ Treg inhibit T-cell proliferation, CTL, and NK cell activities and promote activated T-cell apoptosis.3 IL-4 may be involved in the suppressive activity of CD4⫹CD25⫹ Treg.6 In summary, our study suggests that Foxp3 expressing CD4⫹CD25⫹ Treg underpin “spontaneous” liver transplant tolerance. The regulatory effects of CD4⫹CD25⫹ Treg in vivo may be mediated, at least in part, by CTLA4, TGF, IL-4, and apoptosis of alloreactive T cells. REFERENCES 1. Qian S, Demetris AJ, Murase N, et al: Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 19:916, 1994 2. Li W, Zheng XX, Kuhr CS, et al: CTLA4 engagement is required for induction of murine liver transplant spontaneous tolerance. Am J Transplant 5:978, 2005 3. Sakaguchi S: Naturally arising Foxp3-expressing CD25⫹CD4⫹ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6:345, 2005 4. Liu H, Hu B, Xu D, et al: CD4⫹CD25⫹ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4. J Immunol 171:5012, 2003 5. Benghiat FS, Graca L, Braun MY, et al: Critical influence of natural regulatory CD25⫹ T cells on the fate of allografts in the absence of immunosuppression. Transplantation 79:648, 2005 6. Maerten P, Shen C, Bullens DM, et al: Effects of interleukin 4 on CD25⫹CD4⫹ regulatory T cell function. J Autoimmun 25:112, 2005
P
REVIOUS FINDINGS suggest that the liver is a tolerogenic organ. Induction of hepatic tolerance is an active process, possibly mediated by regulatory T cells (Treg).1,2 CD4⫹CD25⫹ T cells, expressing Foxp3, have been proposed as an important regulator of both self- and transplantation tolerance.3 The role of these Treg in hepatic tolerance is unknown. Our objective was to characterize Foxp3⫹CD4⫹CD25⫹ Treg in liver allograft recipients and examine the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. MATERIALS AND METHODS Orthotopic liver transplantation was performed from B10 (H2b) to C3H (H2k) mice as described.1 Animals were sacrificed at various times from 5 to 100 days posttransplant for Treg characterization by four-color flow cytometry for Foxp3 and transforming growth
factor (TGF)  protein expression by immunohistochemistry and for tolerance transfer by adoptive transfer of spleen cells (SCs) from long-term liver allograft survivors to the C3H mice, which received B10 heart grafts. Rat anti-mouse CD25 monoclonal
From the Departments of Surgery (W.L., K.C., C.S.K., J.D.R., J.D.P.), and Urology (C.S.K.), Division of Transplantation, University of Washington Medical Center, Seattle, Washington, USA; Department of Medicine (X.X.Z.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; Departments of Medicine and Surgery (Y.L., D.L.P.), University of California San Diego, La Jolla, California, USA; and Department of Surgery (A.W.T.), Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. Address reprint requests to Wei Li, MD, PhD. Division of Transplantation, Department of Surgery, University of Washington Medical Center, Box 356174, 1959 NE Pacific Street, Seattle, Washington 98195. E-mail: [email protected]
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.10.093
Transplantation Proceedings, 38, 3205–3206 (2006)
3205
3206 antibody (mAb; PC61; Chimerigen, Allston, Mass, USA; 250 g, intraperitoneally) was administered to liver recipients on days ⫺6, ⫺4, ⫺2 pre- or days 0, 1, 2, 3 posttransplant, respectively. Controls received rat immunoglobulin G (IgG) (250 g; Sigma, Milwaukee, Wis, USA) using an identical regimen. Hamster anti-mouse CTLA4 mAb (UC10-4F10-11) provided by Dr X.X. Zheng (Beth Israel Deaconess Medical Center, Boston, Mass, USA) was administered (250 g, intraperitoneally) on days 0 and 2 to the anti-CD25 mAb pretreated recipients. The following parameters were examined at day 5 posttransplantation: T-cell proliferative activity by MLR; CTL and natural killer (NK) activities by 4hr 51Cr release assay; cytokine interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-␥ production by Elispot; and apoptotic activity by TUNEL staining. Graft survival was analyzed using Kaplan-Maier and others were made using Students t test.
RESULTS
The percentage of CD4⫹CD25⫹ T cells within the CD3 cell population was increased in both liver grafts and recipient spleens posttransplantation compared to syngeneic control or naïve mice. Levels peaked within first 2 weeks. These CD4⫹CD25⫹ T cells expressed intracellular Foxp3 (⬎90%) and CTLA4 (⬎80%). The Foxp3 protein expression detected in frozen sections of liver grafts and host spleens was increased posttransplant from day 5 up to day 100, while TGF production was increased in the spleens posttransplant throughout the time courses compared to naïve mice. Adoptive transfer of the SCs from long-term liver allograft survivors to naïve C3H significantly prolonged B10 heart graft survival [median survival time (MST) ⬎30 days vs control 10 days, P ⬍ .001]. Depletion of recipient CD4⫹CD25⫹ Treg by administration of anti-CD25 mAb, either pre- (MST 14.9 days, n ⫽ 8, P ⬍ .01) or posttransplantation (MST 16.8 days, n ⫽ 6, P ⬍ .01), induced liver allograft acute rejection compared to IgG control. Moreover, CTLA4 blockade by anti-CTLA mAb prevented liver allograft spontaneous acceptance2 and exacerbated liver allograft rejection in anti-CD25 pretreated recipients (MST 7.8 days, n ⫽ 5, P ⬍ .05). In association with liver allograft acute rejection by depletion of CD4⫹CD25⫹ Treg, donor-specific proliferative responses of T cells and anti-donor CTL and NK activities of liver graft infiltrating cells and SCs from anti-CD25 mAbtreated recipients were significantly increased compared with those of cells from control mice. Depletion of Treg also decreased the number of IL-4-producing cells, pro-
LI, CARPER, ZHENG ET AL
moted expansion of IL-2-, IFN-␥-, and IL-10-producing cells, and reduced apoptosis of T cells in the liver grafts and recipient spleens at day 5 posttransplantation. DISCUSSION
Our results suggest that CD4⫹CD25⫹ Treg are expanded after liver transplantation and play a key role in inducing or maintaining “spontaneous” liver transplant tolerance. CTLA4 and TGF may be the key factors in Treg-mediated immune suppression.2– 4 Depletion of recipient CD4⫹CD25⫹ Treg by administration of anti-CD25 mAb significantly reduced the survival of liver allografts. This is also confirmed in major histocompatability complex–mismatched heart and skin transplant models.5 Moreover, anti-CD25 mAb administration plus CTLA4 blockade accelerated liver graft rejection compared to anti-CD25 treatment alone, suggesting that CTLA4 may be involved in CD4⫹CD25⫹ Treg functions.3,4 Our findings also provide evidence that CD4⫹CD25⫹ Treg inhibit T-cell proliferation, CTL, and NK cell activities and promote activated T-cell apoptosis.3 IL-4 may be involved in the suppressive activity of CD4⫹CD25⫹ Treg.6 In summary, our study suggests that Foxp3 expressing CD4⫹CD25⫹ Treg underpin “spontaneous” liver transplant tolerance. The regulatory effects of CD4⫹CD25⫹ Treg in vivo may be mediated, at least in part, by CTLA4, TGF, IL-4, and apoptosis of alloreactive T cells. REFERENCES 1. Qian S, Demetris AJ, Murase N, et al: Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology 19:916, 1994 2. Li W, Zheng XX, Kuhr CS, et al: CTLA4 engagement is required for induction of murine liver transplant spontaneous tolerance. Am J Transplant 5:978, 2005 3. Sakaguchi S: Naturally arising Foxp3-expressing CD25⫹CD4⫹ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol 6:345, 2005 4. Liu H, Hu B, Xu D, et al: CD4⫹CD25⫹ regulatory T cells cure murine colitis: the role of IL-10, TGF-beta, and CTLA4. J Immunol 171:5012, 2003 5. Benghiat FS, Graca L, Braun MY, et al: Critical influence of natural regulatory CD25⫹ T cells on the fate of allografts in the absence of immunosuppression. Transplantation 79:648, 2005 6. Maerten P, Shen C, Bullens DM, et al: Effects of interleukin 4 on CD25⫹CD4⫹ regulatory T cell function. J Autoimmun 25:112, 2005