Ziqin Yin

Osteopontin Expressed in Tubular Epithelial Cells Regulates NK Cell-Mediated Kidney Ischemia Reperfusion Injury

Renal ischemia reperfusion injury (IRI) occurs after reduced renal blood flow and is a major cause of acute injury in both native and transplanted kidneys. Studies have shown diverse cell types in both the innate and the adaptive immune systems participate in kidney IRI as dendritic cells, macrophages, neutrophils, B cells, CD4+ NK+ cells, and CD4+ T cells all contribute to this form of injury. Recently, we have found that NK cells induce apoptosis in tubular epithelial cells (TECs) and also contribute to renal IRI. However, the mechanism of NK cell migration and activation during kidney IRI remains unknown. In this study, we have identified that kidney TECs express a high level of osteopontin (OPN) in vitro and in vivo. C57BL/6 OPN-deficient mice have reduced NK cell infiltration with less tissue damage compared with wild-type C57BL/6 mice after ischemia. OPN can directly activate NK cells to mediate TEC apoptotic death and can also regulate chemotaxis of NK cells to TECs. Taken together, our study’s results indicate that OPN expression by TECs is an important factor in initial inflammatory responses that involves NK cells activity in kidney IRI. Inhibiting OPN expression at an early stage of IRI may be protective and preserve kidney function after transplantation.

Natural Killer Cells Mediate Long-term Kidney Allograft Injury.

Background Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell–mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied. Methods A “parent to F1” kidney transplant model was used to study NK cell–mediated transplant rejection. Results The C57BL/6 kidneys were transplanted into fully nephrectomized CB6F1 (C57BL/6 x BALB/c) mice. Serum creatinine levels increased from baseline (18.8 ± 5.0 &mgr;mol/L to 37.2 ± 5.9 &mgr;mol/L, P < 0.001) at 60 days after transplantation. B6Rag−/−-to-CB6F1Rag−/− (B6Rag−/−xBALB/cRag−/−) recipients, which lack T and B cells but retain NK cells, showed similar levels of kidney dysfunction 65 days after transplantation (creatinine, 33.8 ± 7.9 &mgr;mol/L vs 17.5 ± 5.1 &mgr;mol/L in nontransplant Rag−/− mice, P < 0.05). Importantly, depletion of NK cells in Rag1−/− recipients inhibited kidney injury (24.6 ± 5.5 &mgr;mol/L, P < 0.05). Osteopontin, which can activate NK cells to mediate tubular epithelial cell death in vitro, was highly expressed in 60 days kidney grafts. Osteopontin null kidney grafts had reduced injury after transplantation into CB6F1 mice (17.7 ± 3.1 &mgr;mol/L, P < 0.001). Conclusions Collectively, these data demonstrate for the first time that independent of T and B cells, NK cells have a critical role in mediating long-term transplant kidney injury. Specific therapeutic strategies that target NK cells in addition to conventional immunosuppression may be required to attenuate chronic kidney transplant injury.

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T-cell clonal deletion and suppressing NK cell function.

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft‐versus‐host‐disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation‐free condition. Our data indicate that adoptive transfer of donor‐derived T‐cell receptor (TCR) αβ+CD3+CD4–CD8–NK1.1– (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable‐mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third‐party C3H (H‐2k) skin grafts. Adoptive transfer of CD4+ and CD8+ T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T‐cell alloreactive responsiveness was reduced in the DN Treg cell‐treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4+ and CD8+ T cells. Furthermore, DN Treg‐cell treatment suppressed NK cell‐mediated BM rejection in a perforin‐dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable‐mixed chimerism and donor‐specific tolerance in the irradiation‐free regimen.

Serine protease inhibitor-6 inhibits granzyme B-mediated injury of renal tubular cells and promotes renal allograft survival.

Background Protease inhibitor 9 (PI-9) is an intracellular serpin that specifically inhibits granzyme B, a cytotoxic serine protease found in the cytosolic granules of cytotoxic T lymphocytes and natural killer cells. Enhanced cortical expression of PI-9 has been observed in kidney allografts with subclinical rejection, suggesting that the tubular epithelial cell (TEC) expression of this protein may have a protective role and attenuate overt allograft rejection. Methods and Results We demonstrate that TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase after cytokine exposure. Tubular epithelial cell expression of SPI-6 blocks granzyme B–mediated death because TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. The role of SPI-6 was tested in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2b) into nephrectomized recipients (H-2d). SPI-6 null kidney recipients demonstrated reduced renal function at day 8 after transplantation compared to controls (creatinine, 113±23 vs. 28±3 &mgr;mol/L; n=5; P<0.01), consistent with observed tubular injury and extensive mononuclear cell infiltration. Loss of donor kidney SPI-6 shortened graft survival time (20±19 vs. 66±33 days; n=8–10; P<0.001). Conclusion Our data show for the first time that resistance of kidney TEC to cytotoxic T-cell granzyme B–induced death in vitro and in vivo is mediated by the expression of SPI-6. We suggest that SPI-6 is an important endogenous mechanism to prevent rejection injury from perforin or granzyme B effectors and enhanced PI-9 or SPI-6 expressions by TEC may provide protection from diverse forms of inflammatory kidney injury and promote long-term allograft survival.

Necroptosis Is Involved in Cd4+ T Cell-mediated Microvascular Endothelial Cell Death and Chronic Cardiac Allograft Rejection

Background Despite advances in immunosuppressive therapies, the rate of chronic transplant loss remains substantial. Organ injury involves various forms of cell death including apoptosis and necrosis. We now recognize that early injury of cardiac transplants involves a newly described form of programmed necrotic cell death, termed necroptosis. Because this involves receptor-interacting protein (RIP) kinase 1/3, this study aimed to establish the role of RIP3 in chronic cardiac allograft rejection. Methods We used major histocompatibility complex class II mismatched C57BL/6N (H-2b; B6) or B6.RIP3−/− (H-2b; RIP3−/−) mice to B6.C-H-2bm12 (H2-Ab1bm12; bm12) mouse cardiac transplantation. Microvascular endothelial cells (MVEC) were developed from B6 and RIP3−/− cardiac grafts. Result CD4+ T cell–mediated cardiac graft rejection is inhibited using RIP3 deficient donor grafts, with reduced cellular infiltration and vasculopathy compared with wild type cardiac grafts. Alloreactive CD4+ T cell–mediated MVEC death involves TNF&agr;, Fas ligand (FasL) and granzyme B. Although necroptosis and release of danger molecule high-mobility group box 1 are eliminated by the absence of RIP3, CD4+ T cells had attenuated MVEC death through granzyme B and FasL. Conclusions CD4+ T cell–mediated MVEC death involves in TNF&agr;, FasL and granzyme B. Necroptotic cell death and release of the danger molecule may promote inflammatory responses and transplant rejection. Although loss of RIP3 does not eliminate alloimmune responses, chronic graft injury is reduced. RIP3 is an important therapeutic target but additional granzyme and caspases inhibition is required for sufficiently improving long-term graft survival.

Endogenous Expression of SPI-6 (Serpin Protease Inhibitor-6) in Renal Tubular Cells Inhibits Granzyme B Mediated Injury and Promotes Renal Allograft Survival.: Abstract# C1544

C1544 Endogenous Expression of SPI-6 (Serpin Protease Inhibitor-6) in Renal Tubular Cells Inhibits Granzyme B Mediated Injury and Promotes Renal Allograft Survival. A. Lau,1,3 K. Khan,1 K. Shek,1 Z. Yin,1 X. Huang,1 A. Haig,3 W. Liu,1 Z. Zhang,1,2,3 A. Jevnikar.1,2,3 1Matthew Mailing Centre for Translational Transplant Research, London, Canada; 2Medicine, Western University, London, Canada; 3Pathology, Western University, London, Canada. Introduction: Proteinase inhibitor 9 (PI-9) is an intracellular serpin that inhibits Granzyme B (GrB), a serine protease found in the cytosolic granules of CD8+ T and Natural Killer (NK) cells. PI-9 functions to prevent “misdirected” apoptosis in GrB expressing cells. The murine homolog of PI-9 is serpin protease inhibitor 6 (SPI-6). Kidney tubular epithelial cells (TEC) are a principal targets for cytotoxic cells following transplant. Therefore TEC resistance to cell mediated injury may infl uence the graft survival and function. The expression and regulation of SPI-6 in TEC and kidney has not been studied. Methods/Results: We demonstrate TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase following cytokine exposure. TEC expression of SPI-6 blocks granzyme B mediated death as TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. We then tested the role of SPI-6 in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2b) into nephrectomized recipients (H-2d). SPI-6 null kidney recipients had reduced renal function at day 8 post-transplant compared to controls (creatinine: 113+23 vs. 28+3 μmol/L, n=5, P<0.01) consistent with greater tubular injury and extensive mononuclear cell infi ltration. Finally, loss of donor kidney SPI-6 shortened graft survival time (20+19 vs. 66+33 days, n=8-10, P<0.001). Conclusion: Our data shows for the fi rst time that resistance of kidney TEC to cytotoxic T cell, granzyme B induced death is mediated by the expression of SPI6. We suggest SPI-6 is an important endogenous mechanism to prevent rejection injury from perforin/granzyme B effectors and enhanced PI-9/SPI-6 expression by TEC may provide protection from diverse forms of infl ammatory kidney injury and promote long term allograft survival. Abstract# C1545 The Effi cacy of Immunosuppressants Is Critically Affected By the Recipients Age. K. Edtinger, B. Slegtenhorst, H. Rodriguez Cetina Biefer, X. Yang, M. Quante, H. Uehara, A. ElKhal, S. Tullius. Transplant Surgery and Transplant Surgery Research, Brigham and Women’s Hospital, Boston, MA. Elderly patients are the fastest growing population of transplant recipients. Consequences of immunosenescence are broad and signifi cant, however, only poorly designed in organ transplantation. We hypothesized that aging will impact the effi cacy of immunosuppressants. Skin allografts from young (3 mo) DBA donor mice were transplanted into either young or old (3 and 18 mo) C57BL/6 recipients; animals were treated with either CTLA4-Ig (0,2 mg/d, d0,2,4,6) or rapamycin (rapa) (1 mg/kg/d, d0,1,2,4,6,8,10,12,14) and compared to PBS treated controls; alloimmune responses were assessed serially. Median survival times (MST) in the absence of immunosuppression were 7 and 9 d in young and old recipients, resp.(p=0,012). Interestingly, co-stimulatory blockade with CTLA4-Ig resulted in a modest, however, age-independent prolongation of graft survival (MST=10,5 d and 10,5 d, in young and old recipients, resp.). In contrast, rapa treatment demonstrated impressive age-dependent effects: while MST was only marginally extended to 12 d in young recipients, skin graft survival was signifi cantly prolonged in older recipients (MST=17 d, p=0,031). Next, we dissected effector immune responses linked to age and immunosuppression: Subsequent to co-stimulatory blockade, CD4+ T cells declined signifi cantly in young recipients (p=0,027). At the same time, counts of CD4+CD25+Foxp3+ regulatory T cells (Treg) were reduced in an age-independent fashion subsequent to CTLA4-Ig treatment. Rapa, in contrast, demonstrated age-specifi c effi cacies: counts of CD4+ T cells declined far more pronounced in old compared to young recipients (p=0,012 and <0,0001, resp). Of note, mTOR-inhibition increased Tregs in old mice (p=0,028) while counts remained unchanged in young transplant recipients. Moreover, rapa treatment was linked to a signifi cant decline of CD11c+MHC class II+ cells in old recipients (p=0,03). Furthermore, bromdeoxyuridine incorporation assays demonstrated an inhibition of CD4+ T cell proliferation following CTLA4-Ig treatment only in young allograft recipients (p=0,002), while rapa inhibitied CD4+ T cell proliferation predominantly in old mice (p<0,0001). We demonstrate aging related effi cacies of mTOR inhibition and co-stimulatory blockade. These results are of critical clinical signifi cance and suggest the necessity of age-specifi c adaption of the immunosuppressive regimen. C1545 The Effi cacy of Immunosuppressants Is Critically Affected By the Recipients Age. K. Edtinger, B. Slegtenhorst, H. Rodriguez Cetina Biefer, X. Yang, M. Quante, H. Uehara, A. ElKhal, S. Tullius. Transplant Surgery and Transplant Surgery Research, Brigham and Women’s Hospital, Boston, MA. Elderly patients are the fastest growing population of transplant recipients. Consequences of immunosenescence are broad and signifi cant, however, only poorly designed in organ transplantation. We hypothesized that aging will impact the effi cacy of immunosuppressants. Skin allografts from young (3 mo) DBA donor mice were transplanted into either young or old (3 and 18 mo) C57BL/6 recipients; animals were treated with either CTLA4-Ig (0,2 mg/d, d0,2,4,6) or rapamycin (rapa) (1 mg/kg/d, d0,1,2,4,6,8,10,12,14) and compared to PBS treated controls; alloimmune responses were assessed serially. Median survival times (MST) in the absence of immunosuppression were 7 and 9 d in young and old recipients, resp.(p=0,012). Interestingly, co-stimulatory blockade with CTLA4-Ig resulted in a modest, however, age-independent prolongation of graft survival (MST=10,5 d and 10,5 d, in young and old recipients, resp.). In contrast, rapa treatment demonstrated impressive age-dependent effects: while MST was only marginally extended to 12 d in young recipients, skin graft survival was signifi cantly prolonged in older recipients (MST=17 d, p=0,031). Next, we dissected effector immune responses linked to age and immunosuppression: Subsequent to co-stimulatory blockade, CD4+ T cells declined signifi cantly in young recipients (p=0,027). At the same time, counts of CD4+CD25+Foxp3+ regulatory T cells (Treg) were reduced in an age-independent fashion subsequent to CTLA4-Ig treatment. Rapa, in contrast, demonstrated age-specifi c effi cacies: counts of CD4+ T cells declined far more pronounced in old compared to young recipients (p=0,012 and <0,0001, resp). Of note, mTOR-inhibition increased Tregs in old mice (p=0,028) while counts remained unchanged in young transplant recipients. Moreover, rapa treatment was linked to a signifi cant decline of CD11c+MHC class II+ cells in old recipients (p=0,03). Furthermore, bromdeoxyuridine incorporation assays demonstrated an inhibition of CD4+ T cell proliferation following CTLA4-Ig treatment only in young allograft recipients (p=0,002), while rapa inhibitied CD4+ T cell proliferation predominantly in old mice (p<0,0001). We demonstrate aging related effi cacies of mTOR inhibition and co-stimulatory blockade. These results are of critical clinical signifi cance and suggest the necessity of age-specifi c adaption of the immunosuppressive regimen. Abstract# C1546 B-Cell Depletion Results in Activation-Induced Cell Death in the Leftover T Cells Upon Allogeneic Stimulation. M. Tsai, H. Chien, C. Lee, H. Lai. Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan. Background. Effects of B-cell depletion on allogeneic immune responses have not been well characterized. Methods. We employed a B-cell depletion model to study the effects of B-cell depletion on allogeneic responses. B cells in BALB/c splenocytes were labeled and depleted by a magnetic separation system. The BALB/c splenocytes (with B-cell depletion or not) were stimulated with irradiated C57BL/6 splenocytes and analyzed. Results. Mixed lymphocyte cultures of BALB/c splenocytes with fully allogeneic stimulation showed that B-cell depletion elicited proliferative responses as well as cytokine productions of interleukin (IL)-2 and interferon-γ in the leftover T cells. Interestingly, B-cell depletion signifi cantly increased the percentages of annexin V-positive apoptotic T cells at 192 hours [fi gure 1], and the increases in apoptotic cells could be inhibited by anti-IL-2 antibody. Quantitative-PCR demonstrated that B-cell depletion up-regulated Bim and down-regulated the Bcl-2 to Bim relative expression. Conclusion. B-cell depletion induced activation-induced cell death in the leftover T cells upon allogeneic stimulation. IL-2 and the Bcl-2 family proteins played a critical role in the apoptotic processes of the leftover T cells. C1546 B-Cell Depletion Results in Activation-Induced Cell Death in the Leftover T Cells Upon Allogeneic Stimulation. M. Tsai, H. Chien, C. Lee, H. Lai. Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan. Background. Effects of B-cell depletion on allogeneic immune responses have not been well characterized. Methods. We employed a B-cell depletion model to study the effects of B-cell depletion on allogeneic responses. B cells in BALB/c splenocytes were labeled and depleted by a magnetic separation system. The BALB/c splenocytes (with B-cell depletion or not) were stimulated with irradiated C57BL/6 splenocytes and analyzed. Results. Mixed lymphocyte cultures of BALB/c splenocytes with fully allogeneic stimulation showed that B-cell depletion elicited proliferative responses as well as

RENAL TUBULAR CELL (TEC) EXPRESSION OF SPI-6 (SERPIN PROTEASE INHIBITOR-6) IS REQUIRED FOR PROTECTION FROM GRANZYME B MEDIATED EFFECTOR CELL INJURY FOLLOWING TRANSPLANTATION: 2669

K. Shek1, Z. Zhang2, K. Khan3, S. Wang4, A. Lau5, Z. Yin4, W. Liu6, B. Garcia6, B. Singh3, H. Wang7, A.M. Jevnikar8 1Microbiology And Immunology, University Of Western Ontario, London/Ontario/CANADA, 2Medicine, Unuiversity of Western Ontario, London/Ontario/CANADA, 3, University of Western Ontario, London/CANADA, 4Department Of Medicine, Unuiversity of Western Ontario, London/CANADA, 5, Robarts Research Institute, London/ Ontario/CANADA, 6Department Of Pathology, University of Western Ontario, London/CANADA, 7Department Of Surgery, London Health Sciences Center-University Hospital, London/CANADA, 8, Multi-Organ Transplant Program-London Health Sciences Centre, London/ON/ CANADA