Previously, we showed that the eradication of murine hepatic metastatic colon carcinomas was achieved by using a combination therapy with adenovirus-mediated gene delivery of interleukin 12 (IL-12) and anti-4-1BB agonistic antibody. However, the therapeutic efficacy was compromised severely when mice with tumors larger than 8 x 8 mm(2) were treated. In this report, we studied the effect of OX40 costimulation through agonistic anti-OX40 in combination with the IL-12 + anti-4-1BB immunotherapy on primary and memory anti-tumor cytotoxic T lymphocyte responses in a large-tumor setting (8 x 8 to 12 x 12 mm(2)). Tumor-infiltrating leukocytes (TILs) isolated from mice treated with the combination therapy of IL-12, anti-4-1BB, and anti-OX40 showed a significantly higher ex vivo direct cytotoxic T lymphocyte (CTL) activity against parental tumors, as compared with those from mice treated with IL-12 and anti-4-1BB. In vivo depletion of CD4(+)T cells during combination therapy significantly decreased the number of tumor-infiltrating CD8(+)T cells and their cytotoxic activity in mice treated with IL-12 + anti-4-1BB + anti-OX40 combination therapy but not in the group treated with IL-12 + anti-4-1BB, which indicated that in vivo OX40 engagement on CD4(+) T cells led to the higher CTL responses observed in animals treated with IL-12 + anti-4-BB + anti-OX40 combination therapy. Furthermore, the combination therapy of IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in treated mice, as compared with treatment with IL-12 and anti-4-1BB. More importantly, long-term surviving mice from the combination therapy with IL-12, anti-4-1BB, and anti-OX40 exhibited higher memory CTL responses against parental tumor cells. The results demonstrated that OX40 ligation of CD4(+) T cells facilitated the development of primary and memory CTL responses against tumorcells and that coordinated immune activation by IL-12, anti-4-1BB, and anti-OX40 resulted in a significantly higher survival rate in mice with large tumor burdens. Thus, the combination therapy with the adenovirus encoding IL-12 (Adv.mIL-12) + anti-4-1BB + anti-OX40 antibodies may provide a better treatment modality for patients with advanced cancers, often associated with a state of immune suppression or tolerance.

Objective. To test whether immunizing mice with autoantigens closely linked to mixed connective tissue disease (MCTD) could induce an MCTD-like clinical syndrome distinguishable from systemic lupus erythematosus (SLE).Methods. Transgenic and knockout C57BL/6-derived mice were immunized subcutaneously at age 8-12 weeks with U1-70-kd small nuclear RNP (70K) fusion protein along with either Freund's complete adjuvant (CFA) or U1 RNA. After 2 months, mice were killed and analyzed histologically and serologically. Mixed connective tissue disease (MCTD) is a systemic autoimmune syndrome characterized by antibodies to components of the U1 small nuclear RNP (U1 snRNP), whose clinical manifestations partly overlap those seen in systemic lupus erythematosus (SLE) (1). In contrast to lupus, though, major end organ manifestations, including glomerulonephritis (GN), are not typical features of MCTD. Rather, lung disease, an uncommon manifestation of SLE, is the leading cause of death in MCTD (2). While animal models involving anti-RNP autoantibodies have been previously described (3-7), these models have either demonstrated no clinical disease or manifested double-stranded DNA (dsDNA) antibodies and GN, and thus have not been models of the distinctive features of MCTD.Recent evidence has suggested that components of the U1 snRNP complex may participate directly in provoking the anti-RNP responses seen in MCTD. Autoantibodies in MCTD recognize intact and apoptotically modified forms of individual U1 snRNP proteins (8,9), and frequently also recognize structures composed of multiple subunits of the U1 snRNP macromolecule (10). Moreover, anti-RNP antibodies have been found to interact with lung tissue in ways that could mediate MCTD lung pathology (11). In a small series of mice, we have previously found that direct immunization of a Dr.

The pathologic hallmarks of diabetic nephropathy are excess mesangial extracellular matrix (ECM) and mesangial cell proliferation. We previously showed that mesangial cell phenotypic changes play an important role in the pathogenesis of diabetic nephropathy. We concluded that phenotypic changes were present in bone marrow (BM)-derived mesangial cell progenitors, as transplantation of BM from db/db mice, a model of type 2 diabetic nephropathy, transferred the db genotype and a nephropathy phenotype to naive B6 mice recipients. The recipients did not develop diabetes; however, they did develop albuminuria and glomerular lesions mirroring those in the donors (i.e., glomerular hypertrophy, increased ECM, and increased cell number with cell proliferation). We found that matrix metalloproteinase 2 (MMP-2) facilitated invasion of the mesangial cells into ECM and proliferation in vitro. Thus, increased MMP-2 activity in db/db mesangial cell progenitors may partially explain increased mesangial cell repopulation and proliferation in B6 recipients of db/db BM. In summary, BM-derived mesangial cell progenitors may play a crucial role in the development and progression of ECM accumulation and mesangial cell proliferation in this model of diabetic nephropathy in type 2 diabetes.

We have shown that interleukin-12 (IL-12) generated a strong, albeit transient, anti-tumor response, mostly mediated by natural killer (NK) cell. T cell participation, in addition to NK cells, was essential for persistence of the antitumor response. Ligation of 4-1BB, a co-stimulatory receptor expressed on activated T cells, is known to amplify T cellmediated immunity. In this study, we compared the effect of a systemically delivered agonistic anti-4-1BB monoclonal antibody (anti-4-1BB mAb) with intra-tumoral adenoviralmediated gene transfer of the 4-1BB ligand (ADV/4-1BBL) to liver metastases in a syngeneic animal model of breast cancer. Both treatments induced a dramatic regression of

Objective. To explore the role of immune cells in anti-RNP autoimmunity in a murine model of pneumonitis or glomerulonephritis, using adoptive transfer techniques.Methods. Donor mice were immunized with 50 g of U1-70-kd small nuclear RNP fusion protein and 50 g of U1 RNA adjuvant. Whole splenocytes as well as CD4؉ cell and dendritic cell (DC) subsets from the immunized mice were infused into naive syngeneic recipients. Anti-RNP and T cell responses were assessed by immunoblotting, enzyme-linked immunosorbent assay, and flow cytometry. Development of renal or lung disease was assessed by histology and urinalysis.Results. Unfractionated splenocytes from donor mice without proteinuria induced predominantly lung disease in recipients ( Conclusion. These findings indicate that RNP؉ CD4؉ T cells are sufficient to induce anti-RNP autoimmunity, tissue targeting in anti-RNP autoimmunity can be deviated to either a renal or pulmonary phenotype depending on the presence of accessory cells such as myeloid DCs, and DC subsets can play a role in both propagation of autoimmunity and end-organ targeting.