Development of potent class II transactivator gene delivery systems capable of inducing de novo MHC II expression in human cells, in vitro and ex vivo.

Class II transactivator (CIITA) induces transcription of major histocompatibility complex (MHC) II genes and can potentially be used to improve genetic immunotherapies by converting non-immune cells into cells capable of presenting antigens to CD4+ T cells. However, CIITA expression is tightly controlled and it remains unclear whether distinct non-immune cells differ in this transactivator regulation. Here we describe the development of gene delivery systems capable of promoting the efficient CIITA expression in non-immune cell lines and in primary human cells of an ex vivo skin explant model. Different human cell types undergoing CIITA overexpression presented high-level de novo expression of MHC II, validating the delivery systems as suitable tools for the CIITA evaluation as a molecular adjuvant for gene therapies.

A divergent myeloid dendritic cell response at virus set-point predicts disease outcome in SIV-infected rhesus macaques.

BACKGROUND: The mechanism for loss of myeloid dendritic cells (mDCs) from the circulation in HIV-infected individuals and its relationship to disease progression is not understood. METHODS: A longitudinal analysis of the mDC response in blood and lymph nodes during the first 12 weeks of infection was performed in a cohort of SIVmac251-infected rhesus macaques with different disease outcomes. RESULTS: Monkeys that rapidly progressed to disease or had long-term stable infection had significant losses or increases, respectively, in blood mDCs that were inversely correlated with virus load at set-point. The loss of mDCs from progressor animals was associated with evidence of an increase in CCR7/CCL19-dependent mDC recruitment to lymph nodes and an increase in mDC apoptosis. CONCLUSIONS: mDC recruitment to and death within inflamed lymph nodes may contribute to disease progression in SIV infection, whereas mobilization without increased recruitment to lymph nodes may promote disease control.

Balb/c EGFP mice are tolerant against immunization utilizing recombinant adenoviral-based vectors encoding EGFP: a novel model for the study of tolerance mechanisms and vaccine efficacy.

Enhanced green fluorescent protein (EGFP) is a marker gene product which is readily detectable using the techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. Previous studies have demonstrated the immunogenicity of EGFP in Balb/c mice, identifying an immunodominant H2-K(d) restricted CTL epitope. To model immunological tolerance and vaccine efficiency against self-antigens, we generated a stable transgenic BALB/c mouse expressing EGFP (Balb/c EGFP) through back-crossing C57Bl/6-TG(ACTbEGFP)10sb more than ten times with Balb/c wildtype (wt) mice. High level EGFP expression was detected in the skin and heart, whereas low level expression was observed in the kidney, liver, gut, lung, and spleen. To characterize the immune reactivity to self-antigen, we immunized Balb/c EGFP and Balb/c wt mice with recombinant adenoviral-based vectors encoding EGFP (Ad-EGFP) or beta-galactosidase (Ad-betagal) as a control. Immunization utilizing the Ad-betagal vector expressing 'foreign' antigen induced robust humoral and cellular transgene-specific immunity, whereas Balb/c EGFP mice presented no reactivity following Ad-EGFP immunization against the 'self-antigen' EGFP. These findings describe the creation of a transgenic mouse line tolerant against the common protein marker EGFP, providing a novel system for the evaluation of methods of tolerance disruption and vaccine efficacy.

Dendritic cells: tools and targets for transplant tolerance.

Our knowledge of the role of dendritic cells (DC) in the generation and maintenance of T-cell tolerance has expanded rapidly and is now a key area of research in basic and applied DC biology. This minireview highlights recent developments in the field that are leading to new avenues for exploiting DC in the promotion of transplant tolerance.

Current issues in delivering DCs for immunotherapy.

DC-based immunotherapy has shown early promise in clinical cancer trials, and efforts are being made to determine the optimal method of delivery of cells to achieve the best outcome. While it is accepted that mature DCs are required for stimulation of tumor-specific immunity, the route and frequency of injection and the optimal number of cells needed for clinical success are issues that are still being debated. To solve these questions controlled clinical trials are needed.

Changes in dendritic cell migration and activation during SIV infection suggest a role in initial viral spread and eventual immunosuppression.

Dendritic cells (DC) serve an essential function in linking the innate and acquired immune responses to antigen. Peripheral DC acquire antigen and migrate to draining lymph nodes, where they localize to the T cell-rich paracortex and function as potent antigen presenting cells. We examined the effects of human immunodeficiency virus (HIV) infection on DC function in vivo using the rhesus macaque/simian immunodeficiency virus (SIV) model. Our data show that during acute SIV infection, Langerhans cell density is reduced in skin and activated DC are increased in proportion in lymph nodes, whereas during AIDS, DC migration from skin and activation within lymph nodes are suppressed. These findings suggest that changes in DC function at different times during the course of infection may serve to promote virus dissemination and persistence: early during infection, DC mobilization may facilitate virus spread to susceptible lymph node T cell populations, whereas depressed DC function during advanced infection could promote generalized immunosuppression.

Dendritic cells acquire antigens from live cells for cross-presentation to CTL.

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 microm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3beta and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.

Immunization of chimpanzees with tumor antigen MUC1 mucin tandem repeat peptide elicits both helper and cytotoxic T-cell responses.

CTLs and antibody responses to the tumor-associated antigen MUC1 mucin can be detected in patients with adenocarcinomas of the breast, pancreas, colon, and ovary. However, neither response is generally effective at controlling disease. Methods to augment immunity to MUC1 are being designed, with the expectation that this will lead to an antitumor response. The key to eliciting potent immunity to tumor MUC1 may be in generating MUC1-specific T-helper cell responses, which, to date, have not been reported in cancer patients. We have recently demonstrated that a synthetic vaccine representing five copies of the MUC1 tandem repeat peptide can be used to prime MUC1-specific human CD4+ T cells in vitro. Here, we extend these studies to test the immunogenicity and safety of the tandem repeat peptide in the chimpanzee, which has the identical MUC1 tandem repeat sequence to the human. To promote induction of Th1-type responses, we used the novel adjuvant LeIF, a Leishmania-derived protein that is known to stimulate human peripheral blood mononuclear cells (PBMCs) and antigen-presenting cells, to produce a Th1-type cytokine profile. We found that MUC1 tandem repeat peptide administered with LeIF elicited positive, albeit transient, proliferative T-cell responses to MUC1 in the PBMCs from four of four chimpanzees. Immunization induced MUC1-specific IFN-gamma but not interleukin 4 expression in CD4+ T cells from PBMCs and draining lymph nodes. MUC1-specific CTLs were also generated that did not induce detectable autoimmune dysfunction during the 1 year of observation. We conclude that the MUC1 tandem repeat peptide can be used to elicit both T-helper and cytotoxic cell responses to MUC1 in the primate and holds promise as a safe and effective cancer vaccine.

Retroviral expression of MUC-1 human tumor antigen with intact repeat structure and capacity to elicit immunity in vivo.

MUC-1 mucin is an epithelial cell antigen whose aberrant expression plays a role in autoimmunity and tumor immunity and is thus an attractive candidate for immunotherapy of gene therapy. Because the MUC-1 cDNA is composed almost entirely of 60-bp tandem repeats and is susceptible to homologous recombination, it presents a special challenge to cloning and expression in viral vectors. Nevertheless, we have been successful in constructing a retroviral vector (MFG-MUC-1) with a 22-tandem repeat MUC-1 cDNA. Both stable and transient packaging cell lines are capable of producing high-titer retroviruses that can transfer the expression of MUC-1 to murine 3T3 cells. Transduced cells express uniformly high levels of MUC-1 on their surface, and western blot analysis reveals that the molecule expressed is of full length and extensively glycosylated. We have used the MFG-MUC-1 vector to stably transduce an immortalized murine dendritic cell line and show that immunization of mice with transduced cells elicits specific immune responses to mucin. The ability of this vector to transfer expression of the MUC-1 tumor antigen to potent antigen-presenting cells is expected to be of use in the immunotherapy of epithelial cancers.

Chimpanzee dendritic cells derived in vitro from blood monocytes and pulsed with antigen elicit specific immune responses in vivo.

Dendritic cells differentiated in vitro from blood and other sources using cytokines hold particular promise as immunotherapeutic agents in cancer. However, there are currently no data to show that human in vitro-derived dendritic cells are immunogenic in vivo. We have developed a primate model of immunotherapy using dendritic cells differentiated in vitro from blood monocytes by culturing with human granulocyte/ macrophage colony-stimulating factor and interleukin-4. We measured the immune response to antigen elicited by in vitro-derived and antigen-treated dendritic cells following a single intravenous inoculation an boost in chimpanzees. The antigens tested were ovalbumin, a complex foreign protein, and a peptide derived from the MUC-1 mucin tumor antigen, a relatively uncomplex self antigen. Four chimpanzees were immunized either with antigen-pulsed dendritic cells (two animals) or mock-treated dendritic cells (one animal) given intravenously or both antigens given in adjuvant subcutaneously (one animal). Each animal received a boost of both antigens in adjuvant 10 days later. All animals responded with an IgG-mediated humoral response to ovalbumin measured in the serum at day 24. This was associated with a proliferative cellular response to ovalbumin in the inguinal lymph node draining the boost injection. In contrast, antibody responses to mucin peptide were detected in one animal in response to the boost injection, and no T cell proliferative responses to mucin peptide were detected in the draining lymph node of any animal. To determine if the single inoculation of antigen-pulsed dendritic cells elicited any immunity, we measured the T cell response to ovalbumin in blood mononuclear cells harvested prior to the boost. Ovalbumin-specific proliferative responses that were antigen dose dependent were detected in one of two treated animals. In contrast, ovalbumin given with adjuvant and mock-treated dendritic cells induced no response. The three animals inoculated with dendritic cells, either antigen or mock treated, had moderate T cell responses to bovine serum albumin, a constituent of the medium used to culture cells prior to injection. We conclude from these data that in vitro-derived dendritic cells can elicit T cell responses to a complex foreign antigen following a single intravenous injection in a large primate. It is likely that immunity to a simple self antigen, MUC-1 mucin peptide, may require multiple inoculations. The results support the use of dendritic cells differentiated in vitro as vehicles for immunotherapy in humans.

In vivo migration of dendritic cells differentiated in vitro: a chimpanzee model.

Dendritic cells with potent Ag-presenting function can be propagated from peripheral blood using recombinant cytokines, and these cells have potential usefulness as immunotherapeutic agents in the treatment of cancer and other disease states. However, it is not known if these in vitro differentiated dendritic cells have the capacity to migrate in vivo, especially to T cell areas of lymphoid tissue. We have used a fluorescent marker system to track the migration of dendritic cells, propagated in vitro from chimpanzee peripheral blood, following s.c. injection. We report that injected dendritic cells migrate spontaneously and rapidly to draining lymph nodes, where they remain for at least 5 days. The injected cells interdigitate with T cells in the parafollicular and paracortical zones and retain high level expression of CD86, CD40, and MHC class II molecules, reflecting a phenotype of potent APC. We conclude that dendritic cells differentiated in vitro from peripheral blood and administered s.c. behave in a manner very similar to endogenous Langerhans cells. These data provide strong experimental support, in a highly relevant large animal model, for the use of in vitro differentiated dendritic cells as vehicles for immunotherapy. More importantly, they show that the s.c. route of injection delivers these APC to sites of T cell activation, a prerequisite for the generation of an effective immune response.

Making the most of mucin: a novel target for tumor immunotherapy.

Throughout this brief review I have emphasized the unique biochemical and immunological properties of MUC-1 mucin that make this tumor-associated antigen a novel, exciting and tangible target for tumor immunotherapy. The tandemly repeating nature of the antigenic epitopes in the mucin polypeptide chain, the under-glycosylation of these epitopes, and the expression of the molecule at the cell surface, are all central to the immune recognition of this antigen and must be acknowledged in the design of MUC-1 vaccines. There are considerable difficulties associated with such a design. Short, immunogenic peptides that associate with MHC class I molecules on the cell surface to induce CTL responses are not useful here; MUC-1 must be expressed at the cell surface and this introduces the numerous problems associated with gene transfer. Furthermore, generating under-glycosylated mucin molecules that resemble tumor-associated antigen is not trivial. Competitive inhibition of glycosylation with PhGalNAc is often incomplete, and merely increasing inhibitor concentration results in cell toxicity. But treating tumors is also not trivial. MUC-1 mucin has many characteristics of the ideal tumor-associated antigen, and our understanding of the unique mechanism of mucin recognition on tumors and the appropriate vaccine designs to target this antigen is now advanced. Transgenic mouse and non-human primate models provide excellent preclinical models to test immunotherapeutic and vaccine strategies, and in vitro studies and early-stage clinical trials in humans provide considerable cause for optimism. The next few years in this field should certainly be productive.

Chimpanzee dendritic cells with potent immunostimulatory function can be propagated from peripheral blood.

We have established dendritic cell (DC) cultures from chimpanzee peripheral blood mononuclear cells (PBMC) by using recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh interleukin-4 (IL-4) and demonstrate that these cells have all the characteristics of DC as described for other species. We consistently can obtain 1 x 10(7) DC per 100 ml of blood, a yield of 5% DC as compared to 0.1 to 0.5% DC reported in fresh human PBMC. The cultured DC have a varied morphology with typical cytoplasmic extensions. Phenotypically, the blood-derived DC lack expression of most lineage antigens, but express CD83, an antigen specifically expressed on human blood DC. Chimpanzee DC express very high levels of major histocompatability complex class II antigens, adhesion and costimulatory molecules. Consistent with this phenotype of a powerful antigen-presenting cell, chimpanzee DC generate allogeneic mixed leukocyte responses 15 to 20 times more potent than that elicited by macrophages, Epstein-Barr virus-transformed lymphoblasts and fresh PBMC. In addition, chimpanzee DC very efficiently present tetanus toxoid to PBMC-derived CD4+ T cells as compared to macrophages and PBMC. The DC generated by culturing chimpanzee PBMC with rhGM-CSF and rhIL-4 thus closely resemble human blood-derived DC propagated in the same manner. This technology provides a powerful animal model with which to apply DC to clinical studies with relevance to human disease. In particular, chimpanzee DC can be tested as immunotherapeutic agents for cancer, and be studied in relation to the pathogenesis of human immunodeficiency virus (HIV) infection.

MUC-1 epithelial tumor mucin-based immunity and cancer vaccines.

Many obstacles still stand in the way to eliciting an effective immune response against cancer, even though several antigens and antigenic peptides have been identified as potential tumor targets. All of them, including the MUC-1 mucin, share the caveat of being normal cellular proteins. Unlike all the others, however, MUC-1 expressed on tumors can still be considered a truly tumor-specific antigen. Its expression on normal cells is hidden from the immune system, and its aberrant glycosylation on tumors creates new epitopes recognized by the immune system. Moreover, all other tumor targets identified so far are MHC-restricted peptides that can only be recognized by patients who carry a specific HLA type, or on tumors which continue to express particular HLA alleles. MUC-1 is powerfully different. Recognized as a native molecule independent of MHC, it is a universal immunogen and a universal target, and if made effectively immunogenic, it would be expected to elicit immune responses in all patients, and against numerous MUC-1 expressing human tumors. It may, in fact, be the extraordinary solution to an extraordinary problem of cancer immunity and immunotherapy.

Response of the regional lymph node to bluetongue virus infection in calves.

Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia. In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized. Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN. Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested. In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation. Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies. There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN. In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI. Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8+ T cells. In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population. Double label analysis characterized lymphoblasts as activated CD8+ cells, as determined by expression of MHC Class II antigens (CD8+ MHC II+). These cells were only transiently present as CD8+ MHC II+ cells were not identified in lymph from the challenged LN at 14 DAI. Few CD8+ MHC II+ cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf. The data indicate that B cell proliferation in the challenged LN and release of activated CD8+ cells from this LN were specific responses to BTV infection. The rapid expansion of activated CD8+ T cells indicates that these cells may limit early viral spread. It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

Dynamics of viral spread in bluetongue virus infected calves.

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for 35 to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with interleukin 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of viremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.

Treatment of smoke inhalation in five horses.

Five horses were admitted for treatment of smoke-inhalation injuries sustained in a barn fire. Three of the horses were mildly affected, with high respiratory rates (24 to 36 breaths/min) and normal to low arterial oxygen tensions (77.0 to 94.1 mm of Hg), and responded well to administration of diuretics, bronchodilators, corticosteroids, and antibiotics. The 2 remaining horses were severely affected. Both were in respiratory distress, with markedly low arterial oxygen tensions (50.4 and 57.1 mm of Hg) and cyanosis. These 2 horses required fluid resuscitation in addition to the treatments given to the less severely affected horses. Tracheostomy was performed to facilitate removal of large, obstructive, pseudomembranous tracheobronchial casts. Oxygen was administered by nasal or tracheal insufflation or by use of a high-frequency jet ventilator. The most severely affected horse developed hemorrhagic colitis and was euthanatized. The 4 surviving horses recovered in 2 to 5 months and resumed working without reduction in performance capability.