A Single Amino Acid Substitution Prevents Recognition of a Dominant Human Aquaporin-4 Determinant in the Context of HLA-DRB1*03:01 by a Murine TCR.

BACKGROUND: Aquaporin 4 (AQP4) is considered a putative autoantigen in patients with Neuromyelitis optica (NMO), an autoinflammatory disorder of the central nervous system (CNS). HLA haplotype analyses of patients with NMO suggest a positive association with HLA-DRB1* 03:01. We previously showed that the human (h) AQP4 peptide 281-300 is the dominant immunogenic determinant of hAQP4 in the context of HLA-DRB1*03:01. This immunogenic peptide stimulates a strong Th1 and Th17 immune response. AQP4281-300-specific encephalitogenic CD4+ T cells should initiate CNS inflammation that results in a clinical phenotype in HLA-DRB1*03:01 transgenic mice. METHODS: Controlled study with humanized experimental animals. HLA-DRB1*03:01 transgenic mice were immunized with hAQP4281-300, or whole-length hAQP4 protein emulsified in complete Freund's adjuvant. Humoral immune responses to both antigens were assessed longitudinally. In vivo T cell frequencies were assessed by tetramer staining. Mice were followed clinically, and the anterior visual pathway was tested by pupillometry. CNS tissue was examined histologically post-mortem. Flow cytometry was utilized for MHC binding assays and to immunophenotype T cells, and T cell frequencies were determined by ELISpot assay. RESULTS: Immunization with hAQP4281-300 resulted in an in vivo expansion of antigen-specific CD4+ T cells, and an immunoglobulin isotype switch. HLA-DRB1*03:01 TG mice actively immunized with hAQP4281-300, or with whole-length hAQP4 protein were resistant to developing a neurological disease that resembles NMO. Experimental mice show no histological evidence of CNS inflammation, nor change in pupillary responses. Subsequent analysis reveals that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290 prevents the recognition of hAQP4281-300 by the murine T cell receptor (TCR). CONCLUSION: Induction of a CNS inflammatory autoimmune disorder by active immunization of HLA-DRB1*03:01 TG mice with human hAQP4281-300 will be complex due to a single amino acid substitution. The pathogenic role of T cells in this disorder remains critical despite these observations.

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-γ and tumor necrosis factor-α 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-γ and tumor necrosis factor-α. 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.

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 µg/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.

A short pulse of IL-4 delivered by DCs electroporated with modified mRNA can both prevent and treat autoimmune diabetes in NOD mice.

Bone marrow-derived dendritic cells (DCs) are cells of the immune system that have been used as a tool to boost, modulate, or dampen immune responses. In the context of autoimmunity, DCs can be modified to express immunoregulatory products encoded by transgenes, and used therapeutically in adoptive cellular therapy. DCs that were lentivirally transduced (lt) to express interleukin 4 (IL-4) can significantly delay or prevent the onset of autoimmune diabetes in nonobese diabetic (NOD) mice. However, modifying cells using viral vectors carries the dual risk of oncogenicity or immunogenicity. This study demonstrates that NOD DCs, electroporated with "translationally enhanced" IL-4 mRNA (eDC/IL-4), can be equally efficient therapeutically, despite the reduced amount and shorter duration of IL-4 secretion. Moreover, a single injection of eDC/IL-4 in NOD mice shortly after the onset of hyperglycemia was able to maintain stable glycemia for up to several months in a significant fraction of treated mice. Treatment with eDC/IL-4 boosted regulatory T (Tregs) cell functions and modulated T helper responses to reduce pathogenicity. Thus, treatment with DCs, electroporated with modified IL-4 mRNA to express IL-4 for up to 24 hours, constitutes a viable cellular therapy approach for the regulation of autoimmune diabetes, as a preferred alternative to the use of viral vectors.

Immunologic activity and safety of autologous HIV RNA-electroporated dendritic cells in HIV-1 infected patients receiving antiretroviral therapy.

Immunogenicity, manufacturing feasibility, and safety of a novel, autologous dendritic cell (DC)-based immunotherapy (AGS-004) was evaluated in ten human immunodeficiency virus type 1 (HIV-1)-infected adults successfully treated with antiretroviral therapy (ART). Personalized AGS-004 was produced from autologous monocyte-derived DCs electroporated with RNA encoding CD40L and HIV antigens (Gag, Vpr, Rev, and Nef) derived from each subjects' pre-ART plasma. Patients received monthly injections of AGS-004 in combination with ART. AGS-004 was produced within a mean of 6 weeks and yielded 4-12 doses/subject Full or partial HIV-specific proliferative immune responses occurred in 7 of 9 evaluable subjects. Responses were specific for the AGS-004 presented HIV antigens and preferentially targeted CD8(+) T cells. Mild adverse events included flu-like symptoms, fatigue, and injection site reactions. No evidence of autoimmunity, changes in viral load, or significant changes in absolute CD4(+) and CD8(+) T cell counts were observed. This pilot study supports the further clinical investigation of AGS-004.

The immunosuppressive properties of the HIV Vpr protein are linked to a single highly conserved residue, R90.

BACKGROUND: A hallmark of AIDS progression is a switch of cytokines from Th1 to Th2 in the plasma of patients. IL-12, a critical Th1 cytokine secreted by antigen presenting cells (APCs) is suppressed by Vpr, implicating it as an important virulence factor. We hypothesize that Vpr protein packaged in the virion may be required for disabling APCs of the first infected mucosal tissues. Consistent with this idea are reports that defects in the C-terminus of Vpr are associated with long-term non-progression. PRINCIPAL FINDINGS: Vpr RNA amplified from various sources was electroporated into monocyte-derived DC and IL-12 levels in supernatants were analyzed. The analysis of previously reported C-terminal Vpr mutations demonstrate that they do not alleviate the block of IL-12 secretion. However, a novel single conservative amino acid substitution, R90K, reverses the IL-12 suppression. Analysis of 1226 Vpr protein sequences demonstrated arginine (R) present at position 90 in 98.8%, with other substitutions at low frequency. Furthermore, none of sequences report lysine (K) in position 90. Vpr clones harboring the reported substitutions in position 90 were studied for their ability to suppress IL-12. Our data demonstrates that none of tested substitutions other than K relieve IL-12 suppression. This suggests a natural selection for sequences which suppress IL-12 secretion by DC and against mutations which relieve such suppression. Further analyses demonstrated that the R90K, as well as deletion of the C-terminus, directs the Vpr protein for rapid degradation. CONCLUSION: This study supports Vpr as an HIV virulence factor during HIV infection and for the first time provides a link between evolutionary conservation of Vpr and its ability to suppress IL-12 secretion by DC. DC activated in the presence of Vpr would be defective in the production of IL-12, thus contributing to the prevailing Th2 cytokine profile associated with progressive HIV disease. These findings should be considered in the design of future immunotherapies that incorporate Vpr as an antigen.

Priming of a novel subset of CD28+ rapidly expanding high-avidity effector memory CTL by post maturation electroporation-CD40L dendritic cells is IL-12 dependent.

Dendritic cell (DC)-based immunotherapeutics must induce robust CTL capable of killing tumor or virally infected cells in vivo. In this study, we show that RNA electroporated post maturation and coelectroporated with CD40L mRNA (post maturation electroporation (PME)-CD40L DC) generate high-avidity CTL in vitro that lyse naturally processed and presented tumor Ag. Unlike cytokine mixture-matured DC which induce predominantly nonproliferative effector memory CD45RA(+) CTL, PME-CD40L DC prime a novel subset of Ag-specific CTL that can be expanded to large numbers upon sequential DC stimulation in vitro. We have defined these cells as rapidly expanding high-avidity (REHA) CTL based on: 1) the maintenance of CD28 expression, 2) production of high levels of IFN-gamma and IL-2 in response to Ag, and 3) the demonstration of high-avidity TCR that exhibit strong cytolytic activity toward limiting amounts of native Ag. We demonstrate that induction of REHA CTL is dependent at least in part on the production of IL-12. Interestingly, neutralization of IL-12 did not effect cytolytic activity of REHA CTL when Ag is not limiting, but did result in lower TCR avidity of Ag-reactive CTL. These results suggest that PME-CD40L DC are uniquely capable of delivering the complex array of signals needed to generate stable CD28(+) REHA CTL, which if generated in vivo may have significant clinical benefit for the treatment of infectious disease and cancer.

Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion.

BACKGROUND: RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion. RESULTS: Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction. CONCLUSION: The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types.

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-gamma 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-gamma 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.

Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection.

BACKGROUND: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS: The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE: This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral loads in the absence of conventional antiretroviral drug therapy.