Don Healey

Induction of kidney allograft tolerance by soluble CD83 associated with prevalence of tolerogenic dendritic cells and indoleamine 2,3-dioxygenase.

Background. Tolerogenic dendritic cells (Tol-DCs) play a critical role in inducing and maintaining tolerance. Recognizing that both T-cell inactivation and activation are contingent on signals provided by DCs and that graft-specific activated T cells are major mediators of transplant rejection, we aimed to create an environment favoring Tol-DCs with a novel reagent, human soluble CD83 (hsCD83). Methods. Life-supporting orthotopic kidney transplantation was performed in a C57BL/6-to-BALB/c mouse model. The study group was treated with hsCD83 (100 &mgr;g/mouse/day, postoperative days −1 to +7, intravenously) and compared with untreated controls. Results. Treatment with hsCD83 achieved kidney allograft tolerance (>100 days), with negligible antidonor antibody detected. In contrast, kidney grafts in untreated recipients demonstrated severe rejection after 35 days, characterized by cellular infiltration, interstitial hemorrhage and edema, and glomerular and tubular necrosis, as well as high antidonor antibody titers. In addition, splenic DCs of tolerant recipients exhibited significantly decreased levels of surface major histocompatibility complex class II, CD40, CD80, and intracellular interleukin-12, as well as reduced allogeneic stimulatory capacity. Adoptive transfer of CD11c+ DCs from tolerant hsCD83-treated animals induced kidney allograft tolerance in syngeneic recipients. Blocking indoleamine 2,3-dioxygenase with 1-methyl-tryptophan (15 mg/mouse/day; gavage) prevented the immunosuppressive effect of hsCD83, abrogating hsCD83-induced Tol-DCs and graft tolerance, and leading to acute kidney graft rejection in 22 days. Conclusion. hsCD83 alone was capable of inducing kidney allograft tolerance through a mechanism involving Tol-DC generation and, at least in part, indoleamine 2,3-dioxygenase activity. Because sCD83 is of human origin, the therapeutic approach used in our mouse transplant model holds significant promise for clinical transplantation.

Cytokine Maturation Followed by CD40L mRNA Electroporation Results in a Clinically Relevant Dendritic Cell Product Capable of Inducing a Potent Proinflammatory CTL Response

Dendritic cells (DC) for the immunotherapy of cancer and infectious disease require the appropriate maturation and activation signals to effectively present antigen to drive a proinflammatory response. Here we present a comparison of 4 different maturation protocols for antigen-encoded mRNA electroporated DC. Two protocols rely on cytokine-induced maturation given either preelectroporation or postelectroporation. In addition to the cytokine treatment, 2 further maturation protocols use coelectroporation of CD40L mRNA, with antigen-encoding RNA, to deliver CD40 signals. There were no significant differences in expression of costimulatory molecules such as CD80, CD83, and CD86 or the levels of expression of major histocompatibility complexes. However, results indicate that delivery of an inflammatory signal that includes interferon-γ before the CD40 signal results in high levels of expression of interleukin-12 that was not seen in the absence of CD40L mRNA. All 4 preparations could induce expansion of primary MART-1–specific CD8+ T cells from healthy donors in vitro, but only the 2 processes receiving CD40L could induce interferon-γ expression by those responder cells. Only DC electroporated with CD40L RNA after delivery of the inflammatory signal (PME-CD40L DC), could drive the long-term expansion of MART-1–reactive cells that displayed a CD28+/CD45RA− effector/memory phenotype with strong cytolytic activity.

Potency of mature CD40L RNA electroporated dendritic cells correlates with IL-12 secretion by tracking multifunctional CD8(+)/CD28(+) cytotoxic T-cell responses in vitro.

Electroporation of mature dendritic cells (DC) with RNA-encoding CD40L greatly enhances the production of interleukin (IL)-12, a proinflammatory cytokine necessary for the induction of T-cell immunity. Results presented herein reveal a correlation between the priming of CD28+ antigen-reactive effector memory cytotoxic T lymphocytes (CTL) displaying 3 or 4 simultaneous effector functions and the quantity of IL-12 produced by postmaturation electroporation-CD40L DC. By using multiparameter flow cytometry, the quantities of IL-12 needed to prime naive antigen-reactive T cells to simultaneously produce interferon-&ggr; and tumor necrosis factor-&agr; in the presence or absence of IL-2 secretion in conjunction with lytic activity defined by CD107a expression can be used to determine the overall potency of a DC product. In the presence of IL-12, CTL differentiation toward lytic function is not accompanied by a reduction in the secretion of interferon-&ggr; and tumor necrosis factor-&agr;. Therefore, by measuring the availability of IL-12 one can predict the potency of a DC immunotherapeutics in relation to its ability to drive distinct effector memory CTL subsets with multifunctional activities.

Prevention of chronic renal allograft rejection by soluble CD83.

Background. Recombinant human soluble CD83 had previously exhibited significant immunosuppressive properties that involved interference with dendritic cell maturation in both mouse and humans, inhibition of autoimmunity in mice, and induction of antigen-specific mouse cardiac allograft tolerance when used in combination with other immunosuppressive drugs. Our current research focus turned to examining the effects of peritransplant soluble CD83 (sCD83) administration on prevention of chronic renal allograft rejection. Methods. Fisher344-to-Lewis orthotopic rat renal transplants were performed with sequential recipient killing on postoperative days (PODs) 2, 14, and 140 to examine both the acute and chronic effects of peritransplant sCD83 treatment in rat recipients. Results. Recipients treated with sCD83 exhibited a marked decrease in IgM and IgG deposition in the graft and antidonor antibody levels in the circulation, as early as POD14 and persisting until POD140. sCD83 treatment also reduced the infiltration of T cells and monocytes into the graft tissue and inhibited intragraft expression of MyD88 and inflammatory cytokine levels during the observation period. sCD83-treated grafts demonstrated normal histology beyond POD140, including dramatic reductions in tubular atrophy and interstitial fibrosis compared with untreated recipients. Conclusion. We have demonstrated that peritransplant treatment with recombinant sCD83 attenuates both innate and adaptive immune responses and leads to prevention of chronic rejection in a rat renal transplant model. Because sCD83 is of human origin, the therapeutic approach used in our rodent transplant model holds significant promise for clinical transplantation.

SUPPRESSION OF ANTIDONOR ANTIBODY RESPONSE BY SOLUBLE CD83 PREVENTS CHRONIC RENAL ALLOGRAFT REJECTION: 368

Z. Lan1, W. Ge2, J. Arp3, W. Liu4, S. Brand5, D. Healey6, C. Nicolette5, B. Garcia4, H. Wang3 1Department Of Surgery, London Health Sciences Center-University Hospital , London/ON/CANADA, 2Department Of Surgery, University of Western Ontario, London/ON/CANADA, 3Department Of Surgery, London Health Sciences Center-University Hospital, London/ CANADA, 4Department Of Pathology, University of Western Ontario, London/CANADA, 5, Argos Therapeutics, Durham/NC/UNITED STATES OF AMERICA, 6, 4Argos Therapeutics, Durham/UNITED STATES OF AMERICA