Xuening Hong

Primate renal transplants using immunotoxin.

BACKGROUND T-lymphocyte depletion 7 days before transplantation with immunotoxin FN 18-CRM9 has resulted in tolerance to subsequent renal allografts. We tested the effect of giving immunotoxin on the day of the transplantation and evaluated its effect on rhesus monkey and allograft survival, on antibody production, and on T-cell recovery. METHODS Major histocompatibility complex mismatched renal allografts were performed in rhesus monkeys. Immunotoxin was given starting on the day of transplantation, with and without prednisone and mycophenolate mofetil for 3 days. T-cell subsets and alloantibody levels were measured by flow cytometry. The ability of treated monkeys to develop antibody to tetanus, diphtheria, and xenoantibody was measured. Histology of renal transplants was read in a blinded manner. RESULTS Immunotoxin started on the day of transplantation resulted in prolonged allograft survival in all treatment groups. Graft loss between days 50 and 135 was most often due to interstitial nephritis. Later graft loss was due to chronic rejection. Monkeys had intact antibody responses to alloantigen, tetanus, diphtheria, and xenoantibody. Their CD4 cells recovered gradually over 6 months. CONCLUSIONS Immunotoxin reliably prolongs renal allograft survival when started on the day of transplantation, but interstitial nephritis and chronic rejection limit the development of long-term tolerance. T-cell-dependent B-cell responses remain intact after treatment.

Unaltered graft survival and intragraft lymphocytes infiltration in the cardiac allograft of Cxcr3-/- mouse recipients.

Previous studies showed that absence of chemokine receptor Cxcr3 or its blockade prolong mouse cardiac allograft survival. We evaluated the effect of the CXCR3 receptor antagonist MRL‐957 on cardiac allograft survival, and also examined the impact of anti‐CXCR3 mAb in human CXCR3 knock‐in mice. We found only a moderate increase in graft survival (10.5 and 16.6 days, p < 0.05) using either the antagonist or the antibody, respectively, compared to control (8.7 days). We re‐evaluated cardiac allograft survival with two different lines of Cxcr3−/‐ mice. Interestingly, in our hands, neither of the independently derived Cxcr3−/‐ lines showed remarkable prolongation, with mean graft survival of 9.5 and 10.8 days, respectively. There was no difference in the number of infiltrating mononuclear cells, expansion of splenic T cells or IFN‐γ production of alloreactive T cells. Mechanistically, an increased other chemokine receptor fraction in the graft infiltrating CD8 T cells in Cxcr3−/‐ recipients compared to wild‐type recipients suggested compensatory T‐cell trafficking in the absence of Cxcr3. We conclude Cxcr3 may contribute to, but does not govern, leukocyte trafficking in this transplant model.

Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids

BACKGROUND Anti-CD3 immunotoxin (IT), a T-cell-depleting agent, prolongs survival of renal allografts in a rhesus monkey model without the need for long-term immunosuppression. In this study we sought to further prolong allograft survival by giving short-term conventional immunosuppression simultaneous with IT administration. METHODS MHC class II mismatched, juvenile rhesus monkeys were paired as donor and recipient for renal transplantation. Recipients received two to three daily doses of IT starting on the day of transplantation. Additional immunosuppression was given for no more than 60 days. Graft function was monitored by serum creatinine and renal biopsies. Flow cytometry was used to monitor T-cell recovery. RESULTS Graft survival time (GST) in animals receiving IT was prolonged compared with controls with 50% of IT-treated monkeys surviving >100 days. Animals treated with IT plus mycophenolate mofetil (MMF) and steroids had significantly enhanced GST (mean GST, 305 days) compared with those treated with IT alone (mean GST, 94 days). In contrast, addition of cyclosporine or 40-O-[2-Hydroxyethyl]rapamycin did not significantly increase graft survival time. A comparison among animals from all treatment groups with short (<100 days) and long (>100 days) GST demonstrated that those with the shorter GST had a higher blood T-cell count 2 weeks after transplantation. Full recovery of CD4+ T cells required longer than 6 months. CONCLUSIONS A combination with MMF and steroids given for 4 days after renal allograft transplantation significantly increases GST in IT-treated monkeys. We hypothesize that MMF and steroids suppress the initial T-cell activation mediated by IT.

Increased glomerular deposits of von willebrand factor in chronic, but not acute, rejection of primate renal allografts

BACKGROUND In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model. METHODS Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. RESULTS Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. CONCLUSION Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Modulation of alloimmunity to major histocompatibility complex class I by cotransfer of cytokine genes in vivo

Major histocompatibility complex (MHC) class I antigen is a potent stimulus for alloimmune responses and is the principal immunologic target mediating acute cellular rejection of allografts. Using a method of direct in vivo gene transfer of cDNA encoding donor type MHC class I, we showed in a rat model that recipient muscle could express the transferred MHC class I cDNA, resulting in alloimmunization of the recipient. This was most graphically demonstrated by accelerated rejection of cardiac allografts expressing the same MHC class I as encoded by the immunizing cDNA. We now report the use of the particle-mediated gene transfer via a gene gun (Geneva, Middleton, WI, USA) to transfer MHC class I, as well as cytokine gene expression vectors, into rat skin. Compared to intramuscular injection, gene gun transfer to skin resulted in more efficient immunization. Donor-specific cytotoxic T lymphocyte (CTL) responsiveness and antibody levels increased. Furthermore, coexpression of certain cytokine genes with the MHC class I cDNA modulated the immune response. Specifically, coimmunization with IL-10 cDNA abrogated immunity to allo-MHC class I, while coimmunization with GM-CSF cDNA enhanced it. The influence of expression of these genes in skin was demonstrated by alteration of donor cardiac allograft survival. This model is useful for induction and modulation of alloimmune responses and may be used to develop gene therapy strategies to modify them.