Human CD8 transgene regulation of HLA recognition by murine T cells.

A series of human CD8 transgenic (hCD8 Tg) mice with differential expression in the thymus and periphery were produced to investigate CD8 coreceptor regulation of repertoire selection and T cell responses. Expression of hCD8 markedly enhanced responses to both HLA class I molecules and hybrid A2/Kb molecules providing functional evidence for a second interaction site, outside of the alpha 3 domain, which is essential for optimal coreceptor function. Peripheral T cell expression of hCD8 was sufficient to augment responsiveness to HLA class I, as hCD8 Tg mice which lacked thymic expression responded as well as mice expressing hCD8 in the thymus and periphery. Both murine CD8+ and CD4+ T cells expressing hCD8 transgenes exhibited markedly enhanced responses to foreign HLA class I, revealing the ability of T cell receptor repertoires selected on either murine class I or class II to recognize human class I major histocompatibility complex (MHC). In contrast to recognition of foreign class I, thymic expression of hCD8 transgenes was absolutely required to enhance recognition of antigenic peptide restricted by self-HLA class I. Thus, our studies revealed disparate requirements for CD8 coreceptor expression in the thymus for selection of a T cell repertoire responsive to foreign MHC and to antigenic peptides bound to self-MHC, providing a novel demonstration of positive selection that is dependent on human CD8.

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-alpha in human articular chondrocytes in vitro.

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-alpha in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-alpha. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-alpha or a combination of both (at 10ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10ng/ml TNF-alpha for 6 or 24h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-alpha suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-alpha mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1beta expression by TNF-alpha was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-alpha in chondrocytes.

Endogenous IL-10 regulates sepsis-induced thymic apoptosis and improves survival in septic IL-10 null mice.

Recent studies have shown that increased lymphocyte apoptosis contributes to sepsis-induced mortality. Furthermore, studies have demonstrated that IL-10 can suppress lymphocyte apoptosis, in part, by upregulating Bcl-2 expression and interfering with activation induced cell death. We have previously shown that intrathymic delivery of IL-10 with an adenoviral vector in wild-type mice significantly improves outcome to sepsis. Presently, we investigated the role of endogenous IL-10 expression on thymocyte apoptosis and outcome in IL-10 null mice subject to induction of generalized polymicrobial peritonitis via cecal ligation and puncture. Compared to wild-type C57BL/6 mice, IL-10 null mice demonstrated increased mortality and enhanced lymphocyte apoptosis. Intrathymic injection with an adenoviral vector expressing human IL-10 prior to cecal ligation and puncture in IL-10 null mice significantly improved outcome and decreased thymic caspase-3 activity. Furthermore, plasma concentrations of IL-6 were also significantly reduced in IL-10 null mice treated with the IL-10 expressing adenovirus. In contrast, injection of a control adenovirus did not improve outcome in IL-10 null mice, nor was caspase-3 activity reduced. Thus, local thymic expression of IL-10 not only improves outcome but also reduces local tissue apoptosis and caspase-3 activity, and appears to attenuate the systemic proinflammatory cytokine response.

Spleen but not tumor infiltration by dendritic and T cells is increased by intravenous adenovirus-Flt3 ligand injection.

Dendritic cell (DC) expansion is regulated by the hematopoietic growth factor fms-like tyrosine kinase 3 ligand (Flt3L). DCs are critical to the control of tumor growth and metastasis, and there is a positive correlation between intratumoral DC infiltration and clinical outcome. In this report, we first demonstrate that single intravenous (i.v.) injections of adenovirus (Adv)-Flt3L significantly increased splenic dendritic, B, T and natural killer (NK) cell numbers in both normal and mammary tumor-bearing mice. In contrast, the numbers of DCs and T cells infiltrating the tumors were not increased. Consistent with the minimal effect on immune cell infiltration, i.v. Adv-Flt3L injections had no therapeutic activity against orthotopic mammary tumors. In addition, we noted tumor and Adv-Flt3L expansion of Gr1(+)CD11b(+) immature myeloid suppressor cells (IMSCs), which may inhibit the therapeutic efficacy of Adv-Flt3L-expanded DCs.

Adenovirus delivery provides extended interferon-alpha exposure and augments treatment of metastatic carcinoma.

Type I interferons (e.g. IFNalpha2b) have been successfully used to treat a variety of hematological malignancies, but have not been efficacious for treatment of most solid tumors. We tested the hypothesis that delivery of type I interferon utilizing recombinant adenovirus (rAd) vectors may improve treatment efficacy of metastatic carcinomas by providing increased interferon exposure resulting from continuous transgene expression. Treatment of mice with a rAd-vector expressing hybrid-IFN (rAd-IFNalpha2alpha1) inhibited 4T1 mammary carcinoma tumor growth and induced tumor regression in a dose-dependent manner. Moreover, rAd-IFNalpha2alpha1 treatment reduced hepatic and pulmonary metastatic burden. A comparison of local and systemic routes of administration demonstrated that intratumoral delivery of rAd-IFNalpha2alpha1 was sufficient for inhibition of tumor growth. Moreover, it reduced toxicity associated with high-dose systemic IFNalpha2alpha1 exposure. Interestingly, antitumor activity following intratumoral treatment was due, in part, to the immunostimulatory capacity of the rAd vector component. Furthermore, systemic administration of rAd-IFNalpha2alpha1 potentiated the immunotherapeutic effect induced by local intralesional delivery of empty-rAd vector. These results suggest continuous interferon-alpha exposure may provide improved antitumor responses for metastatic carcinomas. Additionally, immunostimulatory responses induced by rAd-IFNalpha2alpha1 may mitigate the immune-evasive tumor microenvironment.

Immunization with wild-type p53 gene sequences coadministered with Flt3 ligand induces an antigen-specific type 1 T-cell response.

We examined the ability of immunization with sequential adenovirus/plasmid DNA vectors expressing human wild-type p53 to stimulate a type 1 T-cell response and induce protection against challenge from a metastatic tumor that expresses mutated murine p53. We found that tumor protection and an antigen (Ag)-specific immune response were enhanced by prior injection of Flt3 ligand (Flt3L) at a dose and schedule that significantly increased dendritic cell (DC) number and frequency. Preliminary studies using enzyme-linked immunospot and Winn assays suggested that Ag-specific CD8 cells, with their significant increase in IFN-gamma-secreting activity (Tc1 cells), were responsible for the tumor protection. The delayed-type hypersensitivity response to p53 was increased in mice immunized with p53 alone or p53 and Flt3L compared with a negative control. In contrast, spleen cells from mice immunized with p53 and Flt3L exhibited a higher Ag-specific proliferative response than mice immunized with p53 alone. The frequencies of Ag-specific IFN-gamma and interleukin (IL)-4-secreting cells were determined using an enzyme-linked immunospot assay, which demonstrated that the frequency of IFN-gamma-secreting cells was significantly higher in mice immunized with p53 and Flt3L than in mice receiving Flt3L, excipient, or p53 treatment alone. In contrast, the frequency of IL-4-secreting cells did not differ significantly among these groups. We also observed an increased frequency of IL-12 and IFN-gamma-secreting cells (but not IL-4 or IL-10) in the spleens of mice immediately after 10 days of Flt3L treatment, which was also the day of p53 priming. This observation supports the likelihood that there are multiple mechanisms of Flt3L adjuvant activity, including expansion of DC and type 1 T-cell number. Overall, these results suggest that immunization with p53 genetic sequences after in vivo expansion of DC, using Flt3L, provides a useful strategy to induce p53-specific, and protective, type 1 T-cell responses.

Reciprocal allogeneic bone marrow transplantation between NOD mice and diabetes-nonsusceptible mice associated with transfer and prevention of autoimmune diabetes.

Reciprocal allogeneic bone marrow transplantations were carried out between diabetes-susceptible nonobese diabetic (NOD) and diabetes-nonsusceptible C57BL/6 or B10.BR/cd mice to examine the role of the immune system and host environment in the development of autoimmune diabetes. Serotyping of lethally irradiated hosts reconstituted with allogeneic bone marrow showed the hematopoietically derived cells to be of donor origin. Our results showed that lethally irradiated NOD mice reconstituted with a B10.BR/cd hematopoietic cell system remained totally free of insulitis, failed to develop diabetes, and thrived to old age. In contrast, lethally irradiated C57BL/6 or B10.BR/cd mice reconstituted with an NOD hematopoietic cell system all developed insulitis, but only approximately 10% progressed to overt diabetes. Direct adoptive transfer of insulitis and diabetes by mature T-lymphocytes apparently was not required; analogous results were obtained when diabetes-nonsusceptible hosts were reconstituted with NOD hematopoietic cells containing T-lymphocytes or devoid of Thy-1+ cells. The difference in frequency for the development of insulitis versus insulitis plus overt diabetes in C57BL/6 and B10.BR/cd mice suggests that the hematopoietically derived immune cells from NOD mice were sufficient to induce anti-islet reactivity but may require the diabetogenic host environment to develop the frequency and severity of diabetes observed in NOD mice.

A phage display system for detection of T cell receptor-antigen interactions.

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.

Cancer gene therapy using plasmid DNA: pharmacokinetic study of DNA following injection in mice.

The fate of plasmid DNA complexed with cationic lipids delivered intravenously in mice was evaluated at selected timepoints up to 6 months postinjection. Blood half-life and tissue distribution of plasmid DNA and potential expression in tissues were examined. Southern blot analyses of blood indicated that intact plasmid DNA was rapidly degraded, with a half-life of less than 5 min for intact plasmid, and was no longer detectable at 1 hr postinjection. Southern analyses of tissue demonstrated that intact DNA was differentially retained in the lung, spleen, liver, heart, kidney, marrow, and muscle up to 24 hr postinjection. After 7 days, no intact plasmid DNA was detectable by Southern blot analysis; however, the plasmid was detectable by the polymerase chain reaction (PCR) in all tissues examined at 7 and 28 days postinjection. At 6 months postinjection, femtogram levels of plasmid were detected only in muscle. Immunohistochemical analyses did not detect encoded protein in the tissues harboring residual plasmid at 1 or 7 days postinjection.

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.

Ex vivo purging by adenoviral p53 gene therapy does not affect NOD-SCID repopulating activity of human CD34+ cells.

Co-incubation of a replication-deficient, recombinant adenovirus carrying the wild-type p53 gene (rAd-p53) and hematopoietic stem cell (HSC) products from patients with breast cancer can significantly reduce tumor cell contamination. Whereas this approach provides a powerful tumor cell purging strategy, potential detrimental effects on the HSC population have not been investigated. The ability of human HSC to reconstitute hematopoiesis in severe combined immunodeficient (SCID) mice and to undergo secondary transplantation provides the only nonclinical measure of self-renewing, stem cell function. The objective of this study was to investigate whether co-incubation with rAd-p53 compromised the SCID repopulating activity (SRA) of HSC. Granulocyte colony-stimulating factor-mobilized human CD34+ cells were co-cultured with rAd-p53 at our targeted clinical dose, and the ability of these cells to establish multilineage hematopoiesis in sublethally irradiated, nonobese diabetic (NOD)-SCID mice was investigated. The persistence of human cells in the mice was investigated by flow cytometry, granulocyte-macrophage colony-forming unit assay, and polymerase chain reaction of human Alu sequences. Further, limiting dilution analysis provided a quantitative comparison between the SRA of CD34+ cells co-incubated with rAd-p53 and control CD34+ cells (no rAd-p53 co-incubation). We conclude that co-incubation with rAd-p53 has little effect on the SRA of HSC.

Successful semiallogeneic and allogeneic bone marrow reconstitution of lethally irradiated adult mice mediated by neonatal spleen cells.

Spleens of fetal/newborn mice less than 3-4 days of age contain a naturally occurring cell population capable of suppressing T-dependent and T-independent immune responses of third-party adult cells both in vitro and in vivo. We have utilized newborn spleen cells to prevent acute graft-versus-host (GVH) disease in lethally irradiated adult hosts reconstituted with semiallogeneic or even allogeneic bone marrow cells. Pretreatment of reconstituting cell populations with newborn spleen cells reduced the incidence of GVH disease from 100% to 20% in semiallogeneic and from 100% to 40% in allogeneic combinations. Long-term-surviving reconstituted hosts proved immunologically unresponsive to both donor and host histocompatibility antigens, yet possessed a fully chimeric lymphoid system responsive to T and B cell mitogens, as well as unrelated third-party alloantigens.

Identification of Her-2/Neu CTL epitopes using double transgenic mice expressing HLA-A2.1 and human CD.8.

The Her-2/neu protooncogene is associated with malignant transformation and aggressive disease. Because of its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. By identifying potential HLA-A2.1-binding peptides from the Her-2/neu sequence, peptides were selected as candidate T-cell epitopes. The immunogenicity of each peptide was evaluated by priming double transgenic mice expressing both the human (hu) CD8 and HLA-A2.1 molecules with synthetic peptides corresponding to these sequences. Because of the lack of interaction between murine CD8 and HLA-A2.1, expression of huCD8 on murine cells facilitates recognition of HLA molecules on human tumor cell lines. This led to the identification of two peptides that elicit an A2-restricted CTL response, one of which has not been previously identified. Both peptide-specific CTL populations were able to specifically lyse A2.1 and Her-2/neu expressing human tumor cells originating from a variety of tissues, demonstrating the utility of this murine model in identifying peptides presented by human cells. However, several Her-2/neu peptides previously reported to be immunogenic for human CTL were found not to be immunogenic in transgenic mice. The basis for these discrepancies is discussed.

Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis, independent of carcinogens, through activation of innate lymphoid cells.

Chronic inflammation has been associated with increased risk for developing gastrointestinal cancer. Interleukin-23 (IL-23) receptor signaling has been correlated with inflammatory bowel disease pathogenesis, as well as promotion of tumor growth. However, little is known about the relative potential for IL-23-directed causality in gut tumorigenesis. We report that IL-23 transgene expression was sufficient to induce rapid (3-4 weeks) de novo development of intestinal adenomas with 100% incidence. Initiation of tumorigenesis was independent of exogenous carcinogens, Helicobacter colonization, or pre-existing tumor-suppressor gene mutations. Tumorigenesis was mediated by Thy1(+)IL-23R(+) innate lymphoid cells (ILC3), in part, through IL-17 responses as tumor development was inhibited in RAG(-/-) × IL-17(-/-) double knockout mice. Remarkably, IL-23 initiation of tumorigenesis by resident ILCs consistently occurred before recruitment of conspicuous inflammatory infiltrates. Our results reveal an explicit role for IL-23-mediated initiation of gut tumorigenesis and implicate a key role for IL-23R(+) ILC3 in the absence of overt cellular infiltrate recruitment.

Dose-dependent improvements in outcome with adenoviral expression of interleukin-10 in a murine model of multisystem organ failure.

Targeted expression of interleukin-10 (IL-10) has been proposed as a means to suppress acute and chronic inflammation. We explored the capacity of targeted adenoviral expression of human or viral IL-10 to improve outcome in a zymosan-induced model of acute lung injury and multisystem organ failure. Intratracheal administration of adenovirus expressing either human or viral IL-10 prior to zymosan administration significantly improved survival at a dose of 10(7) particles (P<0.01), whereas the same recombinant vectors were ineffective at 10(8) particles and increased mortality at 10(9) particles. Improved survival after administration of 10(7) particles of adenovirus expressing viral or human IL-10 was associated with local tissue expression of IL-10 (100-300 pg/g wet wt). In contrast, mortality after administration of 10(9) particles was associated with markedly elevated IL-10 expression, both in the lung (10000-70000 pg/g wet wt) and systemically (1000-3000 pg/ml plasma), with evidence of an exaggerated systemic inflammatory response (plasma IL-6 and TNFalpha). Targeted gene expression of IL-10 can be used to treat acute inflammatory processes, but increased doses resulting in its systemic release are not associated with improvements in outcome, and may actually exacerbate acute inflammatory processes.

In vivo transduction of thymic dendritic cells with adenovirus and its potential use in acute inflammatory diseases.

Dendritic cells (DC) represent a potential target for gene therapy. In their ability to process antigens and present them to T cells, DC have been allocated a unique role as initiators of the immune response in both the innate and acquired immunity. Recent in vitro studies have showed the feasibility of DC transduction with adenoviral recombinants. In cancer therapy, targeting of DC with adenovirus has been proved to be effective in inhibiting tumour growth, as well as in reducing the number of tumour metastases. The aim of our study is to evaluate the feasibility of in vivo transduction of DC in a murine lymphocyte-rich compartment (thymus) as a potential treatment for acute inflammatory diseases. Nearly 50% of the total thymic DC were transduced with a first-generation adenoviral construct following intrathymic injection, and post-transductional inflammation was neglectable. Transduction of thymic cells with adenoviral recombinants was able to induce the expression of an intracellular protein (beta-galactosidase, green fluorescent protein), as well as the secretion of human interleukin-10, within the local compartment. Furthermore, this induction of the latter significantly decreased thymic apoptosis in the applied model of acute bacterial peritonitis (cecal ligation and puncture).

Gene transfer into hematopoietic progenitor cells mediated by an adeno-associated virus vector.

We describe here the transduction of murine hematopoietic progenitor cells with the dominant selectable neomycin drug-resistance (Neo) gene using a recombinant adeno-associated virus (AAV) vector. Successful transformation of progenitor cells to drug resistance was determined to be approximately 1.5% by colony formation in the presence of geneticin sulfate (G-418). The value of AAV as an alternative to the retrovirus vector systems is discussed.

Transcription of retinoic acid receptor genes in transgenic mice increases CD8 T-cell subset.

Retinoic acid (RA), a vitamin A derivative is known to have a number of effects on the immune system such as inducing cytotoxic T-lymphocytes in vivo and inducing the rejection of skin grafts. However, the molecular mechanisms of these actions are unclear. The retinoic acid receptors which belong to the steroid/thyroid receptor superfamily, are known to bind to regulatory elements of certain genes thereby regulating gene transcription. To determine if expression of retinoic acid receptors in vivo under normal physiological conditions is also regulating genes involved in immunological function, we assayed the human retinoic acid receptor gamma gene driven by a T-cell specific lck-promoter in transgenic mice. Using FACS analysis, we showed that mice expressing the RAR gamma-transgene had significantly increased numbers of CD4-/CD8+ cells compared to controls.