Antibody-targeted vaccination to lung dendritic cells generates tissue-resident memory CD8 T cells that are highly protective against influenza virus infection.

Influenza virus gains entry into the body by inhalation and initiates its replication cycle within the lung. The early stage of infection, while the virus is confined to the lung mucosa, provides the ideal window of opportunity for an effective immune response to control the infection. Tissue-resident memory (Trm) CD8 T cells, located in a variety of tissues including the lung, are ideally situated to act during this window and stall the infection. The factors involved in the differentiation of lung Trm cells remain poorly defined. We demonstrate that recognition of antigen presented locally by dendritic cells (DCs) and transforming growth factor-ß (TGFß) signaling are both required. We exploited this knowledge to develop an antibody-targeted vaccination approach to generate lung Trm cells. Delivering antigen exclusively to respiratory DCs results in the development of lung CD8 Trm cells that are highly protective against lethal influenza challenge. Our results describe an effective vaccination strategy that protects against influenza virus infection.

Receptor-specific delivery of protein antigen to dendritic cells by a nanoemulsion formed using top-down non-covalent click self-assembly.

A new class of targeted and immune-evading nanocarrier made using only biological components and facile processes is assembled in a bottom-up fashion. Simple top-down sequential addition of immune-evading or receptor-specific antibody elements conjugated to biosurfactant protein DAMP4 promotes self-assembly at an interface previously formed in the presence of peptide surfactant AM1, leading to a functional display at the interface through non-covalent molecular self-assembly.

Suppressor of cytokine signalling 1 (SOCS1) is a physiological regulator of the asthma response.

BACKGROUND: The molecular determinants of the severity and persistence of allergic asthma remain poorly understood. Suppressor of cytokine signalling 1 (SOCS1) is a negative regulator of IL-4-dependent pathways in vitro and might therefore control T-helper type 2 (Th2) immunity associated traits, such as IgE levels, mucin production, IL-5 and IL-13 induction, and eosinophilic mucosal inflammation, which are implicated in allergic asthma. OBJECTIVE: To investigate the role of SOCS1 in regulating Th2-associated disease traits in a murine sub-chronic aeroallergen-driven asthma model. METHODS: Following sensitization and challenge with ovalbumin (OVA), bronchoalveolar lavage and serum were collected from mice lacking the Socs1 gene on an IFN-gamma null background (Socs1(-/-)Ifngamma(-/-)). The composition of infiltrating cells in the lung, serum IgE and IgG1 levels and cytokine levels were analysed. RESULTS: Serum IgE levels and infiltrating eosinophils were considerably increased in the lungs of OVA-treated Socs1(-/-)Ifngamma(-/-) mice compared with Ifngamma(-/-) and C57BL/6 controls. Expression of the Th2 cytokines, IL-4, IL-5 and IL-13 was increased in CD4+ cells and lung tissue from OVA-treated Socs1(-/-)Ifngamma(-/-) mice. IgE, IL-5 levels and infiltrating eosinophils were also elevated in saline-treated Socs1(-/-)Ifngamma(-/-) mice, suggesting that in the absence of SOCS1, mice are already biased towards a Th2 response. It is at present unclear whether the elevated cytokine levels are sufficient to result in the exacerbated Th2 response to OVA challenge or whether enhanced intra-cellular signalling also contributes. Surprisingly, of the various IL-4/IL-13 responsive genes tested, only Arginase I appeared to be modestly up-regulated in the lungs of OVA-treated Socs1(-/-)Ifngamma(-/-) mice, suggesting that regulation by SOCS1 occurs primarily in haematopoietic cells and not in the airway epithelium. CONCLUSIONS: Together these results indicate that SOCS1 is an important regulator of the Th2 response.

c-Rel regulates interleukin 12 p70 expression in CD8(+) dendritic cells by specifically inducing p35 gene transcription.

Interleukin 12 (IL-12) is a 70-kD proinflammatory cytokine produced by antigen presenting cells that is essential for the induction of T helper type 1 development. It comprises 35-kD (p35) and 40-kD (p40) polypeptides encoded by separate genes that are induced by a range of stimuli that include lipopolysaccharide (LPS), DNA, and CD40 ligand. To date, the regulation of IL-12 expression at the transcriptional level has mainly been examined in macrophages and restricted almost exclusively to the p40 gene. Here we show that in CD8(+) dendritic cells, major producers of IL-12 p70, the Rel/nuclear factor (NF)-kappaB signaling pathway is necessary for the induction of IL-12 in response to microbial stimuli. In contrast to macrophages which require c-Rel for p40 transcription, in CD8(+) dendritic cells, the induced expression of p35 rather than p40 by inactivated Staphylococcus aureus, DNA, or LPS is c-Rel dependent and regulated directly by c-Rel complexes binding to the p35 promoter. This data establishes the IL-12 p35 gene as a new target of c-Rel and shows that the regulation of IL-12 p70 expression at the transcriptional level by Rel/NF-kappaB is controlled through both the p35 and p40 genes in a cell type-specific fashion.

Molecular cloning of a C-type lectin superfamily protein differentially expressed by CD8alpha(-) splenic dendritic cells.

Dendritic cells (DC) are potent antigen presenting cells that activate naive T cells. It is becoming increasingly clear that DC are not a homogeneous cell population, but comprise different subpopulations that differ in ontogeny and function. To further the molecular characterisation of DC, we screened for genes that were differentially expressed amongst DC subsets and could therefore give insight into their varying biological functions. Using Representational Difference Analysis (RDA) we identified a gene (CIRE) that is expressed at higher levels in the myeloid-related CD8alpha(-) DC than in the lymphoid-related CD8alpha(+) DC. CIRE is a 238 amino acid type II membrane protein, of approximately 33 kDa in size, whose extracellular region contains a C-type lectin domain. Northern blot analysis revealed that CIRE is almost exclusively expressed in DC and was not detected in organs such as heart, brain, kidney, liver, and thymus. T cells failed to express message for CIRE, whilst B cells expressed very low levels. These data here further substantiated by Northern blot analysis of 18 cell lines of various origins (myeloid, macrophage, B and T cell) where only one cell line, which was of myeloid origin and could give rise to DC, expressed mRNA for CIRE. Semi-quantitative RT-PCR suggested that CIRE is down-regulated upon activation. CIRE shares 57% identity with human DC-SIGN, a molecule that has been shown to be the ligand of ICAM-3 and that is also a receptor that binds HIV and facilitates trans-infection of T cells.

Molecular cloning of F4/80-like-receptor, a seven-span membrane protein expressed differentially by dendritic cell and monocyte-macrophage subpopulations.

A novel dendritic cell (DC) surface molecule termed F4/80-like-receptor (FIRE) has been selected based on its differential expression between DC subsets. The gene encoding FIRE has been cloned and sequenced, and mAbs specific for FIRE have been produced. FIRE is a seven-transmembrane-spanning molecule with two epidermal growth factor-like domains in the extracellular region. It is a novel member of the epidermal growth factor/transmembrane-7 protein subfamily and shows similarity to the macrophage marker F4/80. FIRE is expressed by CD8- DC, but not by CD8+ DC, and it is down-regulated on DC activation. It is expressed by blood monocytes and by some tissue macrophages, but not by most macrophage cell lines or by lymphoid cells. FIRE is a useful marker of myeloid cells with a DC developmental potential.

Differential effect of CD8(+) and CD8(-) dendritic cells in the stimulation of secondary CD4(+) T cells.

Dendritic cells (DC), in their role in initiation of the adaptive immune response, have been extensively studied for their capacity to interact and stimulate naive T cells. Subsets of mature murine DC isolated directly from the spleen have been shown to differ in their ability to induce proliferative responses in both primary CD4(+) and primary CD8(+) T cells; the myeloid-related CD8alpha(-) DC induce a more intense or prolonged proliferation of naive T cells than do the lymphoid-related DC bearing CD8alpha despite similar expression of MHC and co-stimulatory molecules. Here we examine the interaction of these DC subpopulations with T cells already in the activated or memory state which are known to have greater sensitivity to antigen stimulation and bear receptors with increased capacity for signal transduction. We show that influenza virus-specific CD4(+) T cell clones and splenic T cells from peptide-primed animals proliferated in response to antigen presented by separated splenic CD8(-) DC. In contrast, these T cells showed only weak, if any, proliferation in response to CD8(+) DC despite observable cluster formation in the cultures. The differential between the two DC types in inducing proliferation was even more pronounced than previously seen with primary T cells and did not reflect differential longevity of the DC in culture, altered response kinetics or deviation from IL-2 to IL-4 induction with CD8(+) DC, but was related to the levels of IL-2 induced. The deficiency in the CD8(+) DC was not overcome by using infectious virus rather than synthetic peptide as the antigen source. These results show that lymphoid-related CD8(+) splenic DC, despite their mature phenotype, fail to provide appropriate signals to secondary CD4(+) T cells to sustain their proliferation.

CD30 prevents T-cell responses to non-lymphoid tissues.

Self antigens can induce T-cell tolerance via a mechanism termed cross-tolerance. This involves the transfer of peripheral tissue antigens to professional APC for presentation in the draining lymph nodes. In this site, CD8+ T cells are activated, proliferate, and are slowly deleted by a CD95-dependent mechanism. Prior to their deletion, some activated cells leave the lymph nodes and encounter antigens on peripheral parenchymal tissues. Without functional CD30, these cells proliferate extensively and cause substantial tissue damage. Thus, CD30 limits autoreactivity, acting as a 'brake' on T-cell proliferation after recognition of autoantigens on parenchymal tissues.

Cytokine gene therapy of mesothelioma. Immune and antitumor effects of transfected interleukin-12.

Malignant mesothelioma (MM) is a solid tumor of the mesothelium for which there is no curative treatment. MM appears to be sensitive to immunotherapeutic approaches, and one of the most powerful immunomodulatory cytokines with antitumor effects is interleukin (IL)-12. We have previously shown in a murine model of MM that systemic administration of recombinant IL-12 induces a potent anti-MM immune response. The nature and accessibility of MM tumors means that they are suitable candidates for direct cytokine and gene-transfer therapeutic approaches. Therefore, we undertook a study to assess the antitumor effects induced by the local production of IL-12 within MM tumors by transfecting a murine MM line with the genes for IL-12. The IL-12 transfectant (AB1-IL-12) did not produce tumors in normal mice, but did so in athymic nude mice, implicating T cells in the prevention of MM tumor growth. In mixing experiments, paracrine IL-12 production inhibited growth of untransfected MM cells provided that cells producing IL-12 represented more than 50-80% of the inoculum. Furthermore, BALB/c mice previously challenged with AB1-IL-12 were protected against rechallenge with parental AB1 tumor, indicating that the transfectant induced long-term immunity. AB1-IL-12 induced systemic immunity that was effective at reducing the incidence of parental AB1 tumor at a distal site, but its effects were dose-dependent. Though both CD4(+) and CD8(+) cells infiltrated the rejecting tumor, CD8(+) effector cells were essential for protection against development of parental AB1 tumor. This study shows that paracrine secretion of IL-12, generated by gene transfer, can induce immunity against MM that can act locally and also at a distant site. In addition, there was no evidence of toxicity, which has been associated with the systemic administration of IL-12, indicating that this cytokine is a good candidate for experimental gene therapy in MM.

Interleukin-12 induces an effective antitumor response in malignant mesothelioma.

Malignant mesothelioma (MM) is a fatal solid tumor of the mesothelium for which there is currently no ameliorating treatment. Using our murine model of this malignancy, which closely resembles the human disease, we have shown that immunotherapy may be of value in the treatment of MM. Because recombinant interleukin-12 (rIL-12) has strong immunomodulatory effects in vivo, we studied the effects of rIL-12 on murine antitumor immune responses, using a nonimmunogenic murine MM tumor cell line (AB1) in vivo. Systemic administration of rIL-12 at the time of tumor inoculation prevented AB1 tumor growth in up to 70% of treated mice, 50% of which were still resistant to AB1 upon rechallenge, indicating that long-term immunologic antitumor effects had been established. This rIL-12-induced effect was dependent on the involvement of both CD4(+) and CD8(+) but not natural killer (NK) cells. Importantly, treatment of established tumors with intralesional injections of rIL-12 resulted in temporary tumor regression or growth inhibition. This effect was dependent on the continuous presence of rIL-12 and correlated with increased numbers of CD4(+) and CD8(+) cells infiltrating the remaining tumor mass. Effective inhibition of tumor growth also occurred when IL-12 was released within MM tumors by coadministration of MM cells that had been stably transfected with the gene for IL-12. These data indicate that IL-12 has potential in the immunotherapy of MM, through gene transfer or local cytokine administration, provided that significant intratumor levels of IL-12 can be achieved for prolonged periods.

Cytokine gene therapy or infusion as treatment for solid human cancer.

In the induction of tissue-directed immune responses, cytokines tend to be released within the affected tissues. We used two strategies to expose tumor tissues to continuous high levels of cytokines: First, a vaccinia interleukin (IL)2 recombinant was injected directly intratumorally 3-weekly at 10(7) pfus/dose in six patients with the solid tumor malignant mesothelioma (MM). No virus excretion was detectable. At each cycle vaccinia-IL-2 mRNA (SQ [semi-quantitative] reverse transcription polymerase chain reaction) was maximal 24-72 h following injection reduced at 8 days and faded by 21 days. No tumor regression occurred. Second, based on the success of granulocyte macrophage colony-stimulating factor (GM-CSF) in gene transfer experiments, we conducted a study using continuous intratumoral GM-CSF infusion in eight patients with MM using a portable pump at doses of 10 micro/cg/24 h over 8 weeks. Systemic neutrophil agglutination and local catheter-related difficulties occurred. Two patients demonstrated tumor necrosis, one of whom had a marked progressive mononuclear cell infiltration of the tumor associated with a partial response (>50% reduction in tumor area). Murine studies using our MM model in CBA and BALB/C mice have demonstrated that B7-1 and allo-class I transfections induce strong tumor-specific cytotoxic T lymphocyte responses: GM-CSF, IL-12, and IL-2 induced mixed nonspecific plus specific responses, whereas B7-2 and class II transfections were not effective. We conclude that increased intratumoral cytokine concentrations can be generated using both gene transfer and cytokine infusion approaches; however, both have their limitations and, at this stage, have not produced dramatic antitumor effects in humans.