Kexing Fan

Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin.

There is an urgent need for improved therapy for advanced ovarian carcinoma, which may be met by administering immune-modulatory monoclonal antibodies (mAbs) to generate a tumor-destructive immune response. Using the ID8 mouse ovarian cancer model, we investigated the therapeutic efficacy of various mAb combinations in mice with intraperitoneal (i.p.) tumor established by transplanting 3 × 106 ID8 cells 10 days previously. While most of the tested mAbs were ineffective when given individually or together, the data confirm our previous finding that 2 i.p. injections of a combination of anti-CD137 with anti-PD-1 mAbs doubles overall survival. Mice treated with this mAb combination have a significantly increased frequency and total number of CD8+ T cells both in the peritoneal lavage and spleens, and these cells are functional as demonstrated by antigen-specific cytolytic activity and IFN-γ production. While administration of anti-CD137 mAb as a single agent similarly increases CD8+ T cells, these have no functional activity, which may be attributed to up-regulation of co-inhibitory PD-1 and TIM-3 molecules induced by CD137. Addition of the anti-cancer drug cisplatin to the 2 mAb combination increased overall survival >90 days (and was probably curative) by a mechanism which included a systemic CD8+ T cell response with tumor specificity and immunological memory. Strikingly, combined treatment of cisplatin and CD137/PD-1 mAb also gave rise to the long-term survival of mice with established TC1 lung tumors. A similar combination of the 2 mAbs and cisplatin should be considered for clinical ‘translation’.

Treatment of collagen-induced arthritis with an anti-osteopontin monoclonal antibody through promotion of apoptosis of both murine and human activated T cells

OBJECTIVE To test the effects of a novel monoclonal antibody (mAb) against human osteopontin (OPN) in the prevention and treatment of collagen-induced arthritis (CIA) and to elucidate the underlying mechanisms of these effects. METHODS DBA/1J mice immunized with type II collagen to induce CIA were monitored to assess the effects of anti-OPN mAb on the clinical severity of the disease, and pathologic changes in the joints were examined histologically. The effects of anti-OPN mAb on survival of activated T cells from arthritic mice and from the synovial fluid of patients with rheumatoid arthritis (RA) were determined by TUNEL assay or annexin V assay. The levels of apoptosis-related proteins (Bim, Bax, and Bcl-2) and NF-kappaB were detected by immunoblot analysis. RESULTS One anti-OPN mAb, 23C3, was effective in inhibiting the development of CIA and even reversing established disease in DBA/1J mice. Monoclonal antibody 23C3 reduced the levels of serum type II collagen-specific autoantibodies and proinflammatory cytokines, and suppressed T cell recall responses to type II collagen. Mechanistic studies demonstrated that OPN prevented the death of type II collagen-activated murine T cells and synovial T cells from RA patients. Monoclonal antibody 23C3 promoted apoptosis of the activated T cells, particularly CD4+ T cells, by inhibiting activation of NF-kappaB and by altering the balance among the proapoptotic proteins Bim and Bax and the antiapoptotic protein Bcl-2. Screening of a phage display peptide library led to identification of the epitope ATWLNPDPSQKQ as being recognized by this novel antibody. CONCLUSION Because of its ability to effectively promote apoptosis of activated T cells, mAb 23C3 may be a novel therapeutic agent for the treatment of RA.

Cimetidine suppresses lung tumor growth in mice through proapoptosis of myeloid-derived suppressor cells

Cimetidine, a histamine type-2 receptor antagonist, is known to inhibit the growth of several tumors in human and animals, however the mechanism of action underlying this effect remains largely unknown. Here, in the mice model of 3LL lung tumor, cimetidine showed significant inhibition of tumor growth. However, an in vitro study demonstrated that cimetidine showed no effect on proliferation, survival, migration and invasion of 3LL cells. We found that cimetidine reduced CD11b(+)Gr-1(+) myeloid derived-suppressive cell (MDSC) accumulation in spleen, blood and tumor tissue of tumor-bearing mice. In vitro coculture assay showed that cimetidine reversed MDSC-mediated T-cell suppression, and improved IFN-γ production. Further investigation demonstrated that the NO production and arginase I expression of MDSCs were reduced, and MDSCs prone to apoptosis by cimetidine treatment. However, MDSC differentiation was not affect by cimetidine. Importantly, although histamine H2 receptor was expressed in MDSC surface, histamine could not reverse the proapoptosis of cimetidine. Moreover, famotidine also did not have this capacity. We found that cimetidine could induce Fas and FasL expression in MDSC surface, and sequentially regulate caspase-dependent apoptosis pathway. Thus, these findings revealed a novel mechanism for cimetidine to inhibit tumor via modulation of MDSC apoptosis.

Myeloid-Derived Suppressor Cells Regulate Immune Response in Patients with Chronic Hepatitis B Virus Infection through PD-1–Induced IL-10

Although myeloid-derived suppressor cells (MDSCs) are well known for their immunosuppressive function in several pathological conditions, the role of MDSCs in hepatitis B virus infection remains obscure. In this study, we investigated the frequency and function of MDSCs in the peripheral blood and liver of 91 chronic hepatitis B (CHB) patients. A higher percentage of MDSCs, defined as CD14+HLA-DR−/low, was detected in peripheral blood of CHB patients than that of the healthy controls. Moreover, high expression of programmed death 1 (PD-1) and secretion of IL-10 in this population were determined. The frequency of MDSCs was positively correlated with serum viral load, but it was negatively correlated with liver inflammatory injury. These cells were also abundant in liver tissue of CHB patients and were related to necroinflammatory activity. Furthermore, we found that these cells could suppress hepatitis B virus–specific CD8+ T cell response, including reduced proliferation and IFN-γ production, and inhibit degranulation of CD8+ T cells, including reduced production of granzyme B and perforin. Importantly, PD-1–induced IL-10 production by MDSCs was responsible for the suppressive activity. To our knowledge, for the first time our study proved that CD14+HLA-DR–/lowPD-1+ MDSCs in CHB patients contribute to an inadequate immune response against the virus and lead to chronic infection, which represents a potential target for therapeutic intervention.

Identification of a novel functional domain of ricin responsible for its potent toxicity

Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved α-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an α-helix comprising the residues 99–106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the α-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the α-helix were rationally designed. Our data clearly demonstrated that the flexibility of the α-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved α-helix might be considered as a potential target for the prevention and treatment of RIP poisoning.

Blockade of LIGHT/HVEM and B7/CD28 signaling facilitates long-term islet graft survival with development of allospecific tolerance.

Background. Previous studies have shown that blockade of LIGHT, a T-cell costimulatory molecule belonging to the tumor necrosis factor (TNF) superfamily, by soluble lymphotoxin β receptor-Ig (LTβR-Ig) inhibited the development of graft-versus-host disease. The cardiac allografts were significantly prolonged in LIGHT deficient mice. No data are yet available regarding the role of the LIGHT/HVEM pathway in more stringent fully allogeneic models such as skin and islet transplantation models. Methods. Streptozotocin-induced chemical diabetic BALB/C mice underwent transplantation with allogeneic C57BL/6 islets and were treated with LTβR-Ig, CTLA4-Ig or a combination of both in the early peritransplant period. Results. Administration of CTLA4-Ig or LTβ R-Ig alone only increased graft survival to 55 days and 27 days respectively, whereas simultaneous blockade of both pathways significantly prolonged the islet allograft survival for more than 100 days. Long-term survivors were retransplanted with donor-specific (C57BL/6) islets and the grafted islets remained functional for more than 100 days. All of islet allografts were protected against rejection when the mixtures of 1×106 CD4+ T cells from tolerant mice and islet allografts were cotransplanted under the renal capsule of the naïve BALB/c recipients. Conclusions. These data indicate that: 1) a synergistic effect for prolonged graft survival can be obtained by simultaneously blocking LIGHT and CD28 signaling in the stringent model of islet allotransplantation; 2) development of donor-specific immunological tolerance is associated with the presence of regulatory T-cell activity; and 3) local cotransplantation of the allografts with the regulatory T cells can effectively prevent allograft rejection and induce donor-specific tolerance in lymphocytes-sufficient recipients.

Cancer Immunotherapy Using In vitro Genetically Modified Targeted Dendritic Cells

Modest clinical outcomes of dendritic cell (DC) vaccine trials call for novel strategies. In this study, we have created a chimeric CD40 molecule that incorporates a single chain Fv (scFv) molecule specific for human ErbB2 antigen and fusing to the membrane spanning and cytosolic domains of murine CD40. After adenoviral transfer to bone marrow-derived DC, this chimeric receptor (CR) induced nuclear factor-kappaB (NF-kappaB)-dependent DC activation and effector function when cultured with immobilized ErbB2 protein or ErbB2-positive tumor cells in vitro. In vivo migration assays showed that approximately 40% injected CR-modified DC (scFv-CD40-DC) effectively migrated to ErbB2-positive tumors, where they were activated after ErbB2 antigen stimulation, and sequentially homed into the draining lymph nodes. In murine ErbB2-positive D2F2/E2 breast tumor (BALB/c) and EL4/E2 thymoma (C57BL/6) models, i.v. injection of 1 x 10(6) scFv-CD40-DC significantly inhibited tumor growth and cured established tumors. Importantly, the cured mice treated by injection of scFv-CD40-DC were effective in preventing both ErbB2-positive and parental ErbB2-negative tumor rechallenge. Analysis of the underlying mechanism revealed that i.v. infusion of scFv-CD40-DC elicited tumor-specific CTL responses, and the transfer of CTLs from scFv-CD40-DC-treated mice protected naive mice against a subsequent tumor challenge. These results support the concept that genetic modification of DC with tumor-associated antigen-specific CD40 chimeric receptor might be a useful strategy for treatment of human cancers.

A humanized anti-osteopontin antibody protects from Concanavalin A induced-liver injury in mice.

Osteopontin has been implicated in various inflammatory diseases including rheumatoid arthritis, multiple sclerosis, Crohns disease, and fulminant hepatitis. Increased expression of osteopontin has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-osteopontin antibody in mice. However, rodent antibodies are highly immunogenic in humans and therefore limited in their clinical application. Here, a murine monoclonal antibody 23C3 against human osteopontin, was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. The humanized version of 23C3, denoted as Hu23C3, was shown to possess affinity comparable to that of its parental antibody. Hu23C3 could also inhibit monocyte migration in response to osteopontin in vitro. Furthermore, in vivo data showed that Hu23C3 significantly protects mice from Concanavalin A (Con A) induced-liver injury in association with the reduction of transaminase activities and improvement of liver injury. Mechanistic studies demonstrated that Hu23C3 inhibited T and NKT cell infiltration, and activation of nuclear factor κB (NF-κB) in the liver, resulting in reduction of TNF-α and IFN-γ production. Thus, our data strongly support that the humanized anti-osteopontin antibody, Hu23C3, may have a potential for the treatment of T cell mediated-hepatitis in human.

IL-17A stimulates the progression of giant cell tumors of bone.

Purpose: Giant cell tumors of bone (GCTB) exhibit aggressive bone lytic behavior. Studies have shown that interleukin 17A (IL-17A) is involved pathologic bone resorption in various skeletal disorders. Thus, we have investigated the role of IL-17A in GCTBs. Experimental Design: We evaluated the progression of GCTBs using Campanacci grading and Enneking staging systems in 74 patients with GCTB. The expression of IL-17A and the IL-17A receptor A (IL-17RA) was assessed in GCTB tissues and in both multinucleated giant cells (MNGC) and stromal cells cultured in vitro using immunostaining and reverse transcription PCR (RT-PCR). The effects of IL-17A on the osteolytic activity of the MNGCs and the proliferation of the stromal cells were investigated using the “pit” formation and MTT assays, respectively. The effects of IL-17A on the expression of proosteolytic factors were examined in primary cultured MNGCs and stromal cells using RT-PCR, Western blotting, and gene expression microarrays. Results: In GCTBs, we detected abundant levels of IL-17A, which were associated with tumor extension and grade. IL-17A is predominantly produced by MNGCs, whereas IL-17RA is expressed by both MNGCs and stromal cells in GCTBs. In the MNGCs, the IL-17A increased the mRNA expression of IL-17A and proosteolytic enzymes, and also enhanced osteolytic ability. In the stromal cells, the IL-17A stimulated cellular proliferation and the expression of proosteolytic factors, including RANKL through myc and STAT3, respectively. In addition, IL-17A stimulated in vivo tumor growth and the extent of angiogenesis in GCTBs. Conclusion: IL-17A stimulates the progression of GCTBs and might represent a useful candidate marker for progression and as a therapeutic target for GCTBs. Clin Cancer Res; 19(17); 4697–705. ©2013 AACR.

Clearance of Propionibacterium acnes by kupffer cells is regulated by osteopontin through modulating the expression of p47phox

Osteopontin (OPN) is a cytokine with multiple functions, including the regulation of innate immune response. However, the detailed function and mechanism of OPN in host defense against invaded microorganisms remain unclear. In this report, we revealed that OPN could affect the clearance of Propionibacterium acnes in kupffer cells. In a murine model of P. acnes induced hepatic granuloma, OPN-deficient mice or wild-type (WT) mice treated with anti-OPN mAb exhibited more hepatic granuloma formation than WT mice. Increased infiltration of intrahepatic leukocytes, higher expression of TLRs, and significantly upregulated level of proinflammatory cytokines of liver tissue were observed in OPN-deficient mice after P. acnes challenge. Moreover, in vitro assay showed that kupffer cells isolated from OPN(-/-) mice exhibited impairment in clearance of P. acnes. Kupffer cells isolated from OPN(-/-) mice showed reduced level of NADPH oxidase-mediated reactive oxygen species (ROS) in response to P. acnes, which was regulated by NADPH oxidase subunit p47phox. Further investigation revealed that OPN interaction with αvβ3 integrin activated PI3K and ERK signal pathways, leading to the expression of p47phox. Taken together, these data demonstrated an important role of OPN in enhancing the antimicrobial innate immune response by modulation of bacterium clearance activity in kupffer cells.