Kifah Shahin

Alterations in chemokine receptor CCR5 expression on blood dendritic cells correlate with acute graft-versus-host disease.

Background Dendritic cells (DC) are important in the development of acute graft-versus-host disease (GVHD) after allogeneic hemopoietic cell transplantation (alloHCT). The trafficking of immature DC from blood to GVHD target organs is likely to be regulated by chemokine receptors. Methods We performed flow cytometry to document the expression of chemokine receptors on circulating DC and correlated the findings after alloHCT with occurrence of acute GVHD. Results In normal individuals, plasmacytoid DC (pDC) expressed high levels of CCR5, whereas the major CD16+ myeloid DC subpopulation lacked CCR5. However, its expression on CD16+ cells was induced by culture in allogeneic mixed lymphocyte reaction supernatant, an effect largely mediated by interferon-&ggr;. CCR5 was expressed on a significant proportion of CD16+ DC in 42 alloHCT patients, whereas it was down-regulated on pDC. The maximum percentage of CCR5+CD16+ DC, at any time after transplantation, correlated with acute GVHD, whereas the minimum CCR5+ on pDC showed a similar correlation. Before developing signs of GVHD, the maximum percentage CCR5+CD16+ DC was higher in patients with GVHD grades II to IV than in GVHD grades 0 and I, whereas the minimum percentage CCR5 on pDC was lower in GVHD grades II to IV than in GVHD grades 0 and I. CCR5 levels more than 20.5% on CD16+ myeloid DC and less than 22.6% on CD123+ pDC correlated with subsequent GVHD grades II to IV with high sensitivities and specificities. Conclusions These observations may reflect DC activation and altered homing during the alloimmune response and could allow early diagnosis and therapeutic intervention before the clinical diagnosis of GVHD.

Bone Marrow Graft-Versus-Host Disease in Reduced Intensity Conditioned Major Histocompatibility Complex Matched Murine Allogeneic Hematopoietic Cell Transplantation

Background The majority of clinical allogeneic haemopoietic cell transplants (alloHCT) are currently performed using reduced intensity conditioning (RIC) instead of myeloablative conditioning (MAC). However, most of the murine models for alloHCT and ensuing complications such as Graft versus Host Disease (GVHD) are studied with MAC models while the biology underlying RIC treatment remains incompletely understood. Methods We investigated a new murine model of major histocompatibility complex (MHC)-matched multiple minor histocompatibility antigen mismatched alloHCT, using 10 x 106 bone marrow (BM) cells and 10-15 x 106 splenocytes from C57BL6 (H-2b) donor mice transplanted into BALB.B (H-2b) recipients after optimizing a RIC protocol consisting of 100mg/kg/day fludarabine for 5 days, 60mg/kg/day cyclophosphamide for 2 days, and sub-lethal total body irradiation (TBI). The MAC model consisted of cyclophosphamide 60mg/kg/day for 2 days and TBI of 850cGy. Mice were scored daily for severity of GVHD for 100 days post-transplant. In other groups of mice, tissue samples from RIC alloHCT mice were collected at different time points to assess the pathological and immunological features of GVHD compared to syngeneic RIC or allogeneic MAC transplant models. Results The lowest TBI dose capable of achieving complete donor chimerism with cyclophosphamide and fludarabine treatment in this strain combination was 325cGy. Mice given RIC had a reduced incidence and delayed onset of GVHD and significantly prolonged survival compared to MAC transplanted recipients. Similar to the MAC model, GVHD associated with RIC showed lymphocytic infiltration of portal tracts and damages bile ducts in liver. Gut pathology was less pronounced in RIC GVHD. In contrast, RIC mice with GVHD showed evidence of BM suppression, with anemia, reduced BM cellularity, and profound reduction in BM B cell lymphopoiesis, associated with damage to the endosteal BM niche. This was associated with an increase in BM CD8+ effector T cells in RIC mice, and elevated blood and BM plasma levels of Th1 cytokines. Increasing doses of splenocytes resulted in increased incidence of GVHD in RIC mice, which was dependent on donor CD8+ and CD4+ effector T cells. We have followed the upregulation of activation markers on donor and recipient dendritic cell and T cell populations in spleen and lymph nodes over time after RIC and MAC alloHCT to compare how the conditioning regimes contribute to immune activation. Conclusion We demonstrate that the BM is a major target organ of GVHD driven by CD8+ and CD4+ effector T cells in an informative, clinically relevant, RIC alloHCT mouse model.

Bone Marrow Graft-Versus-Host Disease in Major Histocompatibility Complex-Matched Murine Reduced-Intensity Allogeneic Hemopoietic Cell Transplantation

Background Most clinical allogeneic hemopoietic cell transplants (alloHCT) are now performed using reduced-intensity conditioning (RIC) instead of myeloablative conditioning (MAC); however, the biology underlying this treatment remains incompletely understood. Methods We investigated a murine model of major histocompatibility complex–matched multiple minor histocompatibility antigen–mismatched alloHCT using bone marrow (BM) cells and splenocytes from B6 (H-2b) donor mice transplanted into BALB.B (H-2b) recipients after RIC with fludarabine of 100 mg/kg per day for 5 days, cyclophosphamide of 60 mg/kg per day for 2 days, and total body irradiation (TBI). Results The lowest TBI dose capable of achieving complete donor chimerism in this mouse strain combination was 325 cGy given as a single fraction. Mice that underwent RIC had a reduced incidence and delayed onset of graft-versus-host disease (GVHD) and significantly prolonged survival compared with MAC-transplanted recipients (TBI of 850 cGy plus cyclophosphamide of 60 mg/kg per day for 2 days). Compared with syngeneic controls, RIC mice with GVHD showed evidence of BM suppression, have anemia, reduced BM cellularity, and showed profound reduction in BM B cell lymphopoiesis associated with damage to the endosteal BM niche. This was associated with an increase in BM CD8+ effector T cells in RIC mice and elevated blood and BM plasma levels of T helper1 cytokines. Increasing doses of splenocytes resulted in increased incidence of GVHD in RIC mice. Conclusions We demonstrate that the BM is a major target organ of GVHD in an informative clinically relevant RIC mouse major histocompatibility complex–matched alloHCT model by a process that seems to be driven by CD8+ effector T cells.

The Monoclonal Antibody 3C12C, Targeting CD83 Is a T Cell Sparing, Potential New Immunosuppressive Agent

A42 Immunosuppressive Capacities of Human Renal Tubular Epithelial Cells; a Role for Indoleamine 2,3-Dioxygenase? M. Demmers, C. Baan, M. Roemeling-van Rhijn, T. van den Bosch, M. Hoogduijn, M. Betjes, W. Weimar, A. Rowshani. Internal Medicine, Section Nephrology and Transplantation, Erasmus MC University Medical Center, Rotterdam, Netherlands. Introduction Renal tubular epithelial cells (TECs) are one of the main targets of T cell attack during acute cellular rejection. We hypothesize that TECs modulate the outcome of allo-immunity in a bi-directional way executing immunosuppressive effects and dampening the local infl ammation. Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme inhibiting T-cell proliferation. TECs express cytoplasmic IDO during acute rejection. We studied whether TECs possess immunosuppressive capacities and if IDO might play a role suppressing T-cell alloactivity. Materials and Methods Anti CD3/CD28 activated peripheral blood mononuclear cells were cocultured with IFN-γ/TNF-α activated TECs for 3 days. We analysed CD4+ T-cell and CD8+ T-cell proliferation response in the absence or presence of IDO inhibitor 1-L-MT. Next we analysed early and late apoptosis as increased IDO acitivity is associated with increased apoptosis. Further we examined whether inhibition of T cell proliferation was cell-cell contact dependent using transwell membrane experiments. Results We found that TECs dose-dependently inhibited CD4+ T-cell and CD8+ T-cell proliferation. TEC mRNA analysis and supernatant L-kynurenine showed that activated TECs express IDO mRNA expression and signifi cantly upregulated L-kynureninen, which was signifi cantly downregulated using 1-L-MT. Transwell experiments showed that TEC-mediated immunosuppression is cell-cell contact dependent. Downregulated CD4+ T-cell proliferation was partly recovered after addition of 1-L-MT, while CD8+ T-cell proliferation was not affected by 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4+ T-cells, 1-L-MT abrogated both early and late CD4+ T-cell apoptosis. Discussion Our data show that TECs possess immunosuppressive capacities and inhibit the allo-reactive T cell proliferation that can partly be explained by indoleamine 2,3-dioxygenase immune regulation. Abstract# A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. Abstract# A44 Angiogenin Promotes Cell Survival During Cyclosporine-Induced Endoplasmic Reticulum Stress. I. Mami,1 N. Bovier,1 S. Pezet,1 P. Beaune,1,2 N. Pallet,1,2 E. Thervet.1,3 1INSERM U-775, INSERM, Paris, France; 2Service de Biochimie, Hopital Europeen Georges Pompidou, Paris, France; 3Service de Nephrologie, Hopital Europeen Georges Pompidou, Paris, France. Background Calcineurin inhibitors nephrotoxicity promotes chronic kidney injury, and contributes to chronic allograft nephropathy. We have demonstrated previously that cyclosporine is an ER stress inducer, ER stress mediates its nephrotoxicity. ER stress contributes to kidney disease, and constitutes a progression factor. Recent studies suggest that Angiogenin (ANG), a stress-activated and secreted ribonuclease, cleaves tRNA to generate fragments called tiRNA. These tiRNA contribute to stress-induced translational repression, indicating that ANG and tiRNA help to reprogram protein translation during stress, and are previously unappreciated components of the stress response. The implication of tiRNA in the ER stress-induced translational repression is unknown. Objectives Our hypothesis is that cyclosporine regulates the production and activation of ANG during the Unfolded Protein Response (UPR), the adaptive program activated in response to ER stress, in the kidney epithelium. That ANG promotes cellular adaptation during stress, mediated by tiRNA integrated in the UPR-induced translational repression. The purpose of this study is to characterize the mechanisms of ANG synthesis, cellular localization and biological functions, during ER stress activated by cyclosporine. Results In a model of human epithelial cells, we have demonstrated that ANG expression is induced during ER stress, that ANG production depends on IRE1a, and that ANG expression is regulated by the transcription factor sXBP1 and NF-kB. ER stress promotes a nucleo-cytoplasmic transfert of ANG which localizes in part in stress granules. ANG inhibits ER stress-indu