Biology of E1-deleted adenovirus vectors in nonhuman primate muscle

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.     

CDP7657, an anti-CD40L antibody lacking an Fc domain,inhibits CD40L-dependent immune responses without thrombotic complications: an in vivo study

IntroductionCD40 ligand (CD40L) blockade has demonstrated efficacy in experimental autoimmune models. However, clinical trials of hu5c8, an anti-human CD40L IgG₁ antibody, in systemic lupus erythematosus (SLE) were halted due to an increased incidence of thrombotic events. This study evaluated CDP7657, a high affinity PEGylated monovalent Fab'' anti-CD40L antibody fragment, to assess whether an Fc-deficient molecule retains efficacy while avoiding the increased risk of thrombotic events observed with hu5c8.MethodsThe potency and cross-reactivity of CDP7657 was assessed in in vitro assays employing human and non-human primate leukocytes, and the capacity of different antibody formats to activate platelets in vitro was assessed using aggregometry and dense granule release assays. Given the important role CD40L plays in regulating humoral immunity, in vivo efficacy was assessed by investigating the capacity of Cynomolgus monkeys to generate immune responses to the tetanus toxoid antigen while the potential to induce thrombotic events in vivo was evaluated after repeat dosing of antibodies to Rhesus monkeys. A PEGylated anti-mouse CD40L was generated to assess efficacy in the New Zealand Black/White (NZB/W) mouse model of SLE.ResultsCDP7657 dose-dependently inhibited antigen-specific immune responses to tetanus toxoid in Cynomolgus monkeys, and in contrast to hu5c8, there was no evidence of pulmonary thrombovasculopathy in Rhesus monkeys. Aglycosyl hu5c8, which lacks Fc receptor binding function, also failed to induce thrombotic events in Rhesus monkeys. In vitro experiments confirmed that antibody constructs lacking an Fc, including CDP7657, did not induce human or monkey platelet activation. A PEGylated monovalent Fab'' anti-mouse CD40L antibody also inhibited disease activity in the NZB/W mouse model of SLE after administration using a therapeutic dosing regimen where mice received antibodies only after they had displayed severe proteinuria.ConclusionsThese findings demonstrate for the first time that anti-CD40L antibodies lacking a functional Fc region do not induce thrombotic events in Rhesus monkeys and fail to activate platelets in vitro but, nevertheless retain pharmacological activity and support the investigation of CDP7657 as a potential therapy for systemic lupus erythematosus and other autoimmune diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0757-4) contains supplementary material, which is available to authorized users.     

Adenovirus CD40 ligand gene therapy counteracts immune escape mechanisms in the tumor Microenvironment

Tumors exhibit immune escape properties that promote their survival. These properties include modulation of Ag presentation, secretion of immunosuppressive factors, resistance to apoptosis, and induction of immune deviation, e.g., shifting from Th1- to Th2-type responses. These escape mechanisms have proven to hamper several immunotherapeutic strategies, and efforts need to be taken to revert this situation. We have studied the immunological effects of introducing CD40 ligand (CD40L), a potent dendritic cell activation molecule, into the tumor micromilieu by adenoviral gene transfer. For this purpose, a murine bladder cancer model (MB49) was used in C57BL/6 mice. The MB49 cells are known to induce IL-10 in the tumor environment. IL-10 potently inhibits the maturation of dendritic cells and thereby also the activation of CTLs. In this paper we show that CD40L immunogene therapy suppresses IL-10 and TGF-beta production (2-fold decrease) and induces a typical Th1-type response in the tumor area (200-fold increase in IL-12 production). The antitumor responses obtained were MB49 cell specific, and the cytotoxicity of the stimulated CD8(+) cells could be blocked by IL-10. Adenovirus CD40L therapy was capable of regressing small tumors (five of six animals were tumor free) and inhibiting the progression of larger tumors even in the presence of other escape mechanisms, such as apoptosis resistance. Furthermore, CD40L-transduced MB49 cells promoted the maturation of dendritic cells (2-fold increase in IL-12) independently of IL-10. Our results argue for using adenovirus CD40L gene transfer, alone or in combination with other modalities, for the treatment of Th2-dominated tumors.     

Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues.

First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.     

Robust vaccine-elicited cellular immune responses in breast milk following systemic simian immunodeficiency virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.     

A comparison of cell-mediated immune responses in rhesus macaques housed singly, in pairs, or in groups

A variety of psychosocial factors have been shown to influence immunological responses in laboratory primates. The present investigation examined the effects of social housing condition on cell-mediated immune responses, comparing rhesus macaques (Macaca mulatta) in three housing conditions (single, pair, and group). Subjects included 12 adults of both sexes in each housing condition (N=36). Multiple blood samples (0, 4, 8, and 12 months) were collected for immunological analyses, including lymphocyte subsets, lymphocyte proliferation to pathogens and nonspecific mitogens, natural killer cell activity, and cytokine production. CD4(+) to CD8(+) ratios differed significantly across housing conditions and singly caged subjects had significantly lower CD4(+)/CD8(+) after the 4-month timepoint than did socially housed (pair and group) subjects. CD4(+) to CD8(+) ratios were positively correlated within subjects, suggesting a trait-like aspect to this parameter. Lymphocyte proliferation responses to all four gastrointestinal pathogens differed across housing conditions (at least at the 0.08 level), as did proliferation responses to StaphA, and the production of cytokines (IFN-gamma, IL-2, and IL-10). Proliferation responses of singly caged monkeys did not differ from socially housed monkeys and the highest levels of both IFN-gamma and IL-10 were produced by group housed subjects. The data demonstrate that social housing condition affects immune responses. While not unidirectional, these effects generally suggest enhanced immune responses for socially housed animals. Since rhesus monkeys live socially in nature, and the immune responses of singly housed animals differed from those housed socially, there is considerable motivation and justification for suggesting that the use of singly housed rhesus macaques may complicate interpretations of normal immunological responses. This may have important implications for the management, treatment, and selection of primate subjects for immunological studies.     

Th2-dependent B cell responses in the absence of CD40-CD40 ligand interactions

CD40 is thought to play a central role in T cell-dependent humoral responses through two distinct mechanisms. CD4+ T helper cells are activated via CD40-dependent Ag presentation in which CD80/CD86 provides costimulation through CD28. In addition, engagement of CD40 on B cells provides a direct pathway for activation of humoral responses. We used a model of adenovirus-mediated gene transfer of beta-galactosidase (lacZ) into murine lung to evaluate the specific CD40-dependent pathways required for humoral immunity at mucosal surfaces of the lung. Animals deficient in CD40L failed to develop T and B cell responses to vector. Activation of Th2 cells, which normally requires CD40-dependent stimulation of APCs, was selectively reconstituted in CD40 ligand-deficient mice by systemic administration of an Ab that is agonistic to CD28. Surprisingly, this resulted in the development of a functional humoral response to vector as evidenced by formation of germinal centers and production of antiadenovirus IgG1 and IgA that neutralized and prevented effective readministration of vector. The CD28-dependent B cell response required CD4+ T cells and was mediated via IL-4. These studies indicate that CD40 signals to the B cells are not necessary for CD4+ Th2 cell-dependent humoral responses to be generated.     

Magnitude and phenotype of cellular immune responses elicited by recombinant adenovirus vectors and heterologous prime-boost regimens in rhesus monkeys

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for human immunodeficiency virus type 1 (HIV-1) and other pathogens have been shown to elicit antigen-specific cellular immune responses. Rare serotype rAd vectors have also been constructed to circumvent preexisting anti-Ad5 immunity and to facilitate the development of novel heterologous rAd prime-boost regimens. Here we show that rAd5, rAd26, and rAd48 vectors elicit qualitatively distinct phenotypes of cellular immune responses in rhesus monkeys and can be combined as potent heterologous prime-boost vaccine regimens. While rAd5-Gag induced primarily gamma interferon-positive (IFN-gamma(+)) and IFN-gamma(+)/tumor necrosis factor alpha(+) (TNF-alpha(+)) T-lymphocyte responses, rAd26-Gag and rAd48-Gag induced higher proportions of interleukin-2(+) (IL-2(+)) and polyfunctional IFN-gamma(+)/TNF-alpha(+)/IL-2(+) T-lymphocyte responses. Priming with the rare serotype rAd vectors proved remarkably effective for subsequent boosting with rAd5 vectors. These data demonstrate that the rare serotype rAd vectors elicited T-lymphocyte responses that were phenotypically distinct from those elicited by rAd5 vectors and suggest the functional relevance of polyfunctional CD8(+) and CD4(+) T-lymphocyte responses. Moreover, qualitative differences in cellular immune responses may prove critical in determining the overall potency of heterologous rAd prime-boost regimens.     

Enhanced breadth of CD4 T-cell immunity by DNA prime and adenovirus boost immunization to human immunodeficiency virus Env and Gag immunogens

A variety of gene-based vaccination approaches have been used to enhance the immune response to viral pathogens. Among them, the ability to perform heterologous immunization by priming with DNA and boosting with replication-defective adenoviral (ADV) vectors encoding foreign antigens has proven particularly effective in eliciting enhanced cellular and humoral immunity compared to either agent alone. Because adenoviral vector immunization alone can elicit substantial cellular and humoral immune responses in a shorter period of time, we asked whether the immune response induced by the prime-boost immunization was different from adenoviral vaccines with respect to the potency and breadth of T-cell recognition. While DNA/ADV immunization stimulated the CD8 response, it was directed to the same epitopes in Gag and Env immunogens of human immunodeficiency virus as DNA or ADV alone. In contrast, the CD4 response to these immunogens diversified after DNA/ADV immunization compared to each vector alone. These findings suggest that the diversity of the CD4 immune response is increased by DNA/ADV prime-boost vaccination and that these components work synergistically to enhance T-cell epitope recognition.     

Preexisting immunity to adenovirus in rhesus monkeys fails to prevent vector-induced toxicity

In an earlier study we evaluated innate immune responses to a first-generation adenoviral vector infused into the portal vein of rhesus monkeys who had never been exposed to adenovirus previously. In these animals, the systemic administration of E1/E3-deleted adenoviral vectors resulted in immediate activation of innate immunity and serious toxicity caused by targeting of vector to antigen-presenting cells and systemic inflammation. We analyze here how these responses are affected by vector-specific preexisting immunity that was induced by intramuscular immunization 6 months prior to evaluation. Our results show that preexposure to the vector substantially diminishes the transgene expression in most tissues but has little effect on gene transfer. Significantly, preimmunization does not eliminate systemic vector-induced toxicity. These conclusions are based on the presence of clinical features of coagulopathy and elevated levels of proinflammatory cytokine interleukin-6 in the serum of animals treated with vector after intramuscular immunization. Furthermore, preexisting immunity appears to induce a vector-specific inhibitory effect on erythroid progenitor development in the bone marrow that is not found when naive animals are challenged with vector.     

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 10⁸ PFU) or low-dose (1 × 10⁷ PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates.     

Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates

The magnitude and durability of immune responses induced by replication-defective adenovirus serotype 5 (ADV5) vector-based vaccines were evaluated in the simian-human immunodeficiency virus/rhesus monkey model. A single inoculation of recombinant ADV5 vector constructs induced cellular and humoral immunity, but the rapid generation of neutralizing anti-Ad5 antibodies limited the immunity induced by repeated vector administration. The magnitude and durability of the immune responses elicited by these vaccines were greater when they were delivered as boosting immunogens in plasmid DNA-primed monkeys than when they were used as single-modality immunogens. Therefore, administration of ADV5-based vectors in DNA-primed subjects may be a preferred use of this vaccine modality for generating long-term immune protection.     

免疫共刺激分子OX40L对乙型肝炎核酸疫苗的免疫佐剂作用

[目的]为了进一步增强HBV DNA疫苗的免疫反应,本研究将共刺激分子OX40L 作为HBV DNA疫苗的分子佐剂免疫小鼠,旨在探讨共刺激分子OX40L对HBV DNA疫苗诱导体液和细胞免疫应答的影响.[方法]我们将HBV DNA疫苗(pcDS2)单独或联合共刺激分子质粒pOX40L免疫C57BL/6小鼠;分别在第0,2,4周进行免疫,在第6周检测抗-HBs IgG、IgG1和IgG2a,T淋巴细胞增殖指数,细胞因子表达水平和体内细胞毒性T淋巴细胞杀伤作用(CTL)等免疫学指标.[结果]pceDS2联合pOX40L免疫组小鼠的抗-HBs水平显著提高,抗-HBs IgG亚类以IgG2a占优;免疫小鼠的T淋巴细胞体外经乙型肝炎表面抗原(HBsAg)刺激后,联合免疫组刺激指数(SI)明显高于pcDS2组;联合免疫组CD4 + T淋巴细胞的IL-4和IFN-γ表达水平及CD8 + T淋巴细胞的IFN-γ表达水平显著升高;DNA疫苗免疫的各组小鼠,HBsAg特异性体内CTL高于对照组,其中联合免疫组小鼠的体内CTL杀伤作用最强.[结论]共刺激分子OX40L不仅能增强HBV DNA疫苗诱导特异性体液免疫应答,还能增强特异性细胞免疫反应,尤其增强体内CTL的杀伤活性,为HBV DNA疫苗的研究奠定了基础.     

SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques

Current evidence suggests that a strong induced CD8 human immunodeficiency virus type 1 (HIV-1)-specific cell mediated immune response may be an important aspect of an HIV vaccine. The response rates and the magnitude of the CTL responses induced by current DNA vaccines in humans need to be improved and cellular immune responses to DNA vaccines can be enhanced in mice by co-delivering DNA plasmids expressing immune modulators. Two reported to work well in the mouse systems are interleukin (IL)-12 and CD40L. We sought to compare these molecular adjuvants in a primate model system. The cDNA for macaque IL-12 and CD40L were cloned into DNA vectors. Groups of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag alone or in combination with either IL-12 or CD40L. CD40L did not appear to enhance the cellular immune response to SIVgag antigen. However, more robust results were observed in animals co-injected with the IL-12 molecular adjuvant. The IL-12 expanded antigen-specific IFN-gamma positive effector cells as well as granzyme B production. The vaccine immune responses contained both a CD8 component as well a CD4 component. The adjuvanted DNA vaccines illustrate that IL-12 enhances a CD8 vaccine immune response, however, different cellular profiles.     

Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody

BACKGROUND: CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor (TNF) family, plays a critical role in both humoral and cellular immune responses and has been implicated in biological pathways involving epithelial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L with CD40 receptor expressed on B cells. It results in various B cell responses, including immunoglobulin isotype switching and B cell differentiation and proliferation. These responses can be inhibited by the monoclonal antibody 5c8, which binds with high affinity to CD40L. RESULTS: To understand the structural basis of the inhibition, we determined the crystal structure of the complex of the extracellular domain of CD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that the complex has the shape of a three-bladed propeller with three Fab fragments bound symmetrically to a CD40L homotrimer. To further study the nature of the antibody-antigen interface, we assessed the ability of 23 site-directed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal microscopy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. CONCLUSIONS: The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.     

Elicitation of Both Anti HIV-1 Env Humoral and Cellular Immunities by Replicating Vaccinia Prime Sendai Virus Boost Regimen and Boosting by CD40Lm

For protection from HIV-1 infection, a vaccine should elicit both humoral and cell-mediated immune responses. A novel vaccine regimen and adjuvant that induce high levels of HIV-1 Env-specific T cell and antibody (Ab) responses was developed in this study. The prime-boost regimen that used combinations of replication-competent vaccinia LC16m8Δ (m8Δ) and Sendai virus (SeV) vectors expressing HIV-1 Env efficiently produced both Env-specific CD8⁺ T cells and anti-Env antibodies, including neutralizing antibodies (nAbs). These results sharply contrast with vaccine regimens that prime with an Env expressing plasmid and boost with the m8Δ or SeV vector that mainly elicited cellular immunities. Moreover, co-priming with combinations of m8Δs expressing Env or a membrane-bound human CD40 ligand mutant (CD40Lm) enhanced Env-specific CD8⁺ T cell production, but not anti-Env antibody production. In contrast, priming with an m8Δ that coexpresses CD40Lm and Env elicited more anti-Env Abs with higher avidity, but did not promote T cell responses. These results suggest that the m8Δ prime/SeV boost regimen in conjunction with CD40Lm expression could be used as an immunization platform for driving both potent cellular and humoral immunities against pathogens such as HIV-1.     

Comparative analysis of antitumor activity of CD40L, RANKL, and 4-1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cells

The tumor necrosis factor (TNF) family comprises a group of ligands that regulate cell proliferation, differentiation, activation, maturation and apoptosis through interaction with the corresponding TNF receptor family members. In this study, we have evaluated whether adenovirus-mediated intratumoral gene transfer of CD40L, RANKL, or 4-1BBL elicits an immune response to established murine MC38 and TS/A tumors. Intratumoral administration of the recombinant adenoviral vectors expressing CD40L, RANKL or 4-1BBL 7 days post-tumor cell inoculation resulted in significant inhibition of MC38 tumor growth for all three ligands when compared with control groups treated with either saline or control adenovirus. However, intratumoral injection of Ad-4-1BBL or Ad-CD40L resulted in a significantly stronger inhibition of TS/A tumor progression than did Ad-RANKL treatment. We also demonstrated that intratumoral administration of dendritic cells (DC) transduced with adenoviral vectors encoding the TNF-related ligands resulted in a significant inhibition of MC38 tumor growth as compared with control groups treated with Ad-LacZ-transduced DC or saline-treated DC. In addition, DC overexpressing CD40L secreted considerably more IL-12 and expressed higher levels of the co-stimulatory molecules, CD80, CD86 and CD40, than did DC overexpressing LacZ, 4-1BBL or RANKL. We have also demonstrated that DC/CD40L, DC/4-1BBL, and DC/RANKL survived significantly longer than control DC or DC infected with the LacZ vector. Taken together, these results demonstrate that adenoviral gene transfer of CD40L, RANKL or 4-1BBL elicit a significant antitumor effect in two different tumor models, with CD40L gene transfer inducing the strongest antitumor effect.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

Regulation of CD40L expression on natural killer cells by interleukin-12 and interferon γ: its role in the elicitation of an effective antitumor immune response

Effector cell functions are regulated by a number of positive signals for the mediation of antitumor immunity. The CD40 and CD40 ligand (CD40L) interaction has been implicated in the generation of effective cell-mediated and humoral immune responses, where cytokines have been shown to play a significant role in the expression of these molecules. Our earlier studies have shown that spontaneous regression of a rat histiocytoma transplanted s.c. is mediated by CD8⁺ CD3⁻ NK cells. The CD40-CD40L mediation during tumor regression was of interest. Tumor-transplanted animals showed enhanced expression of CD40L on natural killer (NK) and T cells, when compared to cells from normal animals. CD40 expression on AK-5 tumor cells was also induced after s.c. transplantation. Administration of anti-(interleukin-12) (anti-IL-12) and anti-(interferon γ) (anti-IFNγ) antibodies in tumor-bearing animals showed down-regulation of the expression of CD40L on NK and T cells with simultaneous inhibition of cytotoxic acitivity of NK cells, cytokine release and the production of antitumor antibody. Naive NK cells, when co-cultured with fixed AK-5 cells, were induced to express CD40L. CD40L expression modulated the immune response exerted by NK cells, in part by the activation of nuclear factor kB (NF-kB). Furthermore, the signaling via CD40L through the use of anti-CD40L antibody promoted the in vitro activation of cytotoxic as well as NF-kB binding activity in NK cells from tumor-transplanted animals. These observations demonstrate that the expression of CD40L by the effector cells is regulated by IL-12 and IFNγ, and could effectively modulate the NK-cell-mediated immune response during the regression of AK-5 tumor. Received: 1 June 2000 / Accepted: 27 July 2000     

Repeated administration of adenoviral vectors in lungs of human CD4 transgenic mice treated with a nondepleting CD4 antibody.

The central role of CD4+ T cells in regulation of adenovirus vector-mediated immune responses has been documented previously in murine models. We analyzed the effects of a nondepleting mAb to human CD4 (CD4 mAb; Clenoliximab) on immune functions following intratracheal administration of adenoviral vectors in murine CD4-deficient mice (muCD4KO) expressing a human CD4 transgene (HuCD4 mice). Treatment of HuCD4 mice with Clenoliximab inhibited both cell-mediated and humoral immune responses to adenoviral Ags. Chronic treatment of HuCD4 mice with Clenoliximab permitted successful readministration of adenoviral vectors at least four times. The ability to readminister these vectors is associated with marked suppression of neutralizing Ab responses to viral capsid proteins. Clenoliximab also inhibited CTL and prolonged expression of the transgene. T or B cell responses to adenovirus did not emerge after the effects of a short course of Clenoliximab diminished. These data illustrate the potential utility of a nondepleting CD4 Ab in facilitating gene therapy using adenoviral vectors.