Gene Therapy (2003) 10, 991–998 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt REVIEW Adaptive and innate immune responses to gene transfer vectors: role of cytokines and chemokines in vector function D Chen1, B Murphy2,3, R Sung2 and JS Bromberg1,2 1Carl C Ichan Center for Gene Therapy and Molecular Medicine, New York, NY, USA; 2Recanati/Miller Transplantation Institute, New York, NY, USA; and 3Division of Nephrology, Mount Sinai School of Medicine, New York, NY, USA Gene Therapy (2003) 10, 991–998. doi:10.1038/sj.gt.3302031 Keywords: chemokine; cytokine; innate immunity; gene therapy Introduction motifs. The character of the immune response depends on the type of vector, the route of administration, the The infection, transfection, or transduction of cells, parenchymal cell types involved, and the concurrent tissues, and organs by necessity involves an interaction disease state of the host. Thus, microbial pathogenesis, of gene transfer vectors or gene medicines with the when viewed from this perspective, is not simply the immune system of the recipient. In the simplest terms, immune response to foreign viral protein antigens. this immune interaction is often viewed as a variant of The section on Innate immunity of this review will viral or bacterial pathogenesis. Foreign microbial protein examine chemokines and innate immunity. Recent antigens, the components of the vectors, are taken up by evidence demonstrates powerful stereotyped responses professional or nonprofessional antigen-presenting cells of leukocytes and tissue parenchymal cells to viruses and (APCs), and the antigens presented in the context of host nucleic acids. These responses do not depend on classic major histocompatibility complex (MHC) antigens to T- or B-cell immunity. Among these responses are the CD4+ helper T cells. The activated helper T cells then production of a wide array of potent soluble mediators provide additional stimulatory signals to B cells to make termed chemokines. Other innate immune responses, antibodies, to induce CD8+ cytotoxic T cells, to activate which may lead to chemokine production, include the phagocytic macrophages, or to stimulate other CD4+ T activation of Toll receptors, activation of antigen non- cells. specific leukocytes, and induction of nucleic acid The resulting inflammatory milieu is then responsible sensitive intracellular second messenger pathways. Im- for removing free vector, cell-associated vector, vector- portantly, innate and adaptive immune responses inter- transduced cells, and transgene products. The concep- act, amplify, and regulate each other, which is the focus tual framework then for understanding these immune of the section on Interplay of innate and adaptive responses, so they can be manipulated and thereby immunity of this review. improve the performance of the vectors, has centered The section on Cytokine and chemokine regulation of around dissecting out how particular vectors interact vector function of this review will summarize specific with classical immune responses. Important components mechanisms of how cytokines and chemokines regulate of immunity are the soluble cytokines and chemokines vector function at the level of individual cells and tissues. produced by the multifarious interactions among vec- Recent reports demonstrate that it is not simply immune tors, leukocytes, and parenchymal cells. However, a clearance of vector, transgene product, or transduced study of these soluble mediators shows a far more varied cells that prevents successful gene transfer and expres- and complex series of events than those outlined above, sion; but rather more subtle effects on the pathways of and suggests not only many more levels of immune vector entry, transport, integration, and transcriptional complexity, but also opportunities to intervene in activation that determine transgene expression. Lastly, immune interactions to manipulate vector function. the review will draw conclusions about opportunities for The section on Adaptive immunity of this review will further investigation and directions for improving the focus on adaptive immune responses. Immunity is utility and efficacy of gene medicines. engendered not only in response to specific vector or transgene peptide antigens, but also in response to nonantigenic nucleic acid components, such as CpG Adaptive immunity Correspondence: Dr JS Bromberg, Mount Sinai School of Medicine, Box Adaptive immunity to vector antigens or transgene 1104, One Gustave L Levy Place, New York, NY 10029-6574, USA product can enhance or inhibit the intended therapeutic Adaptive and innate immune responses to gene transfer vectors D Chen et al 992 effect. Antivector immunity can enhance cell killing of inoculation of plasmid DNA into muscle or skin results target tissue or enhance the effectiveness of immuniza- in the activation of plasmid-containing DCs at the tion for gene therapy for infectious diseases. On the other injection site, in draining lymph nodes, and in systemic hand, vector persistence and transgene expression are circulation.16,17 These cells function as APC, can induce limited by antivector immunity, a disadvantage in specific proliferation of CD4+ T cells and generation of transplantation or metabolic diseases. Adenovirus vec- CD8+ CTLs, and can induce both primary and secondary tors are the most immunogenic and also the most adaptive immune responses.15,18 The direct pathway extensively studied of the gene transfer vectors, from appears to be the primary mode of antigen presentation an immunologic perspective; hence most of the discus- in this model, rather than indirect presentation of soluble sion that follows refers primarily to adenovirus. antigen by untransfected DC. Administration of first-generation, E1/E3-deleted ade- DCs infected in vitro with adenovirus encoding an novirus vectors generates well-characterized adaptive antigenic peptide can induce specific CTL responses that immune responses against viral capsid proteins.1,2 The can be augmented with repeated administration of development of cellular and humoral adaptive immunity infected DC.19 In contrast to direct immunization, which appears to be the primary mechanism by which generates neutralizing antibody that may limit repeat transgene expression is extinguished; the cellular arm administration of vector, low titers of neutralizing anti- effecting viral clearance, and the humoral response body are generated with this method. Thus, the capacity preventing effective readministration of vector. Each of of DCs to function as effective APCs may potentially be these immune response is associated with the production utilized for therapeutic benefits in designing vaccines or of a large number of key cytokines that facilitate T-cell– in tumor immunization. APC interactions. Transgene expression is generally Adaptive immunity to gene therapy vectors is tradi- enhanced when reagents or knockout mice, designed to tionally thought of as being primarily because of vector inhibit adaptive immunity, are utilized.3,4 Adeno-asso- antigens and transgene product. However, immunity can ciated virus (AAV) vectors, although less efficient than also be induced by vector nucleic acids. Bacterial DNA is adenovirus vectors, incite much less of an immune a powerful stimulator of polyclonal proliferation in B response. Consequently, expression by these vectors cells and cytokine production by monocytes and lym- results in greater vector persistence and transgene phocytes.20,21 These properties are attributed to un- expression.5 methylated CpG motifs present in plasmid DNA or Adaptive immunity to adenovirus has been attributed synthetic oligonucleotides, motifs which are much less to low levels of viral gene expression, or may reflect frequent in mammalian DNA, which is not immunosti- contamination by wild-type virus. In support of viral mulatory.22 These responses are sensitive to inhibitors of gene expression as a mechanism for antivector immunity endosomal acidification, which is coupled to the genera- is a diminution of Th2 responses and neutralizing tion of intracellular reactive oxygen species and the antibodies by E4-deleted vectors,6 and the absence of degradation of IkB.23,24 Indeed, inhibition of IkB degra- late inflammation in muscle when ‘gutted’ vectors dation suppresses the immunostimulatory effects of CpG lacking all viral genes are employed.7 However, vectors DNA.25 CpG DNA also induces the activation of c-Jun N- rendered biologically inactive can generate similar CD4+ terminal kinase and p38 MAPK, which lead to activation and CD8+ lymphocyte infiltration as do untreated of the transcription factor AP-1.26,27 The actions of CpG vectors, and antivector CTL responses can be generated motifs stimulate the secretion of cytokines such as TNFa in the absence of vector transcription.8,9 In addition, and IL-6, monocyte activation, and NK cell activity.23,28,29 empty adenoviral vectors can serve as potent adjuvants, The induction of type I interferons, IFNg, IL-12, and IL- implying that immunogenicity can be independent of 18, stimulates Th1 responses and enhance the develop- vector function.10 Therefore, cellular responses may not ment of adaptive immunity.30–33 Although CpG motifs necessarily require viral gene expression. Interestingly, are generally immunostimulatory to most cell types, both cellular and humoral immunity can be dramatically evidence exists that different sequences possess different diminished by modification of the vector with capsid cellular profiles