Nuclear-localized costimulatory molecule 4-1BBL promotes colon cancer cell proliferation and migration by regulating nuclear Gsk3ß, and is linked to the poor outcomes associated with colon cancer.

Anti-tumor immune response and the prognosis of tumor are the results of competition between stimulatory and inhibitory checkpoints. Except for upregulating inhibitory checkpoints, lowering some immune accelerating molecules to convert an immunostimulatory microenvironment into an immunodormant one through "decelerating the accelerator" might be another effective immune escape pattern. 4-1BBL is a classical transmembrane costimulatory molecule involving in antitumor immune responses. In contrast, we demonstrated that 4-1BBL is predominantly localized in the nuclei of cancer cells in colon cancer specimens and is positively correlated with tumor size, lymph node metastasis, and a lower survival ratio. Furthermore, the nuclear localization of 4-1BBL was also ascertained in vitro. 4-1BBL knockout (KO) arrests the proliferation and impaired the migration and invasion ability of colon cancer cells in vitro and retarded tumor growth in vivo. 4-1BBL KO increased the accumulation of Gsk3ß in the nuclei of colon cancer cells and consequently decreased the expression of Wnt pathway target genes and thus alter tumor biological behavior. We hypothesized that unlike membrane-expressed 4-1BBL, which stimulates the 4-1BB signaling of antitumor cytotoxic T cells, the nuclear-localized 4-1BBL could facilitate the malignant behavior of colon cancer cells by circumventing antitumor signaling and driving some key oncotropic signal pathway in the nucleus. Nuclear-localized 4-1BBL might be an indicator of colon cancer malignancy and serve as a promising target of immunotherapy.

DNA damage and gene expression changes in patients exposed to low-dose X-radiation during neuro-interventional radiology procedures.

Interventional radiology-based imaging is the preferred choice for diagnosis and therapy of many complex diseases, despite possible adverse effects of the radiation exposures. We have measured induced DNA damage and changes in gene expression in relation to entrance surface dose (ESD) in peripheral blood samples of patients (n = 51) who underwent neuro-interventional radiological procedures. The ESD values, measured by thermoluminescence dosimetry, were 4.9-273 mGy (forehead), 14-398 mGy (eyes), 8-433.3 mGy (shoulders), and 4.7-242.5 mGy (thyroid). The in-built recorded Dose Area Product (DAP) values were 74.61-558.55 and 13.17-2825.12 Gy*cm2 for diagnostic and therapeutic procedures, respectively. The mean fluorescence intensity (MFI) on the phosphorylation of γ-H2AX and p53ser-15 was higher in samples obtained post-exposure vs. pre-exposure. However, the increase was statistically significant only for p53ser-15 (P < 0.01). Consistent with γ-H2AX, CDKN1A, FDXR, BAX, DDB2, SESN1, BCL2, MDM2, TNFSF10B, and PCNA showed (non-significant) decreased expression while GADD45A, ATM, and TNFSF9 showed (non-significant) increased expression. Our results suggest that most of the patients had increased DNA damage and altered gene expression after receiving relatively low doses of ionising radiation. This implies that these procedures should be carried out at the lowest possible doses of radiation that do not compromise image quality.

EOMES-positive CD4+ T cells are increased in PTPN22 (1858T) risk allele carriers.

The presence of the PTPN22 risk allele (1858T) is associated with several autoimmune diseases including rheumatoid arthritis (RA). Despite a number of studies exploring the function of PTPN22 in T cells, the exact impact of the PTPN22 risk allele on T-cell function in humans is still unclear. In this study, using RNA sequencing, we show that, upon TCR-activation, naïve human CD4+ T cells homozygous for the PTPN22 risk allele overexpress a set of genes including CFLAR and 4-1BB, which are important for cytotoxic T-cell differentiation. Moreover, the protein expression of the T-box transcription factor Eomesodermin (EOMES) was increased in T cells from healthy donors homozygous for the PTPN22 risk allele and correlated with a decreased number of naïve CD4+ T cells. There was no difference in the frequency of other CD4+ T-cell subsets (Th1, Th17, Tfh, Treg). Finally, an accumulation of EOMES+ CD4+ T cells was observed in synovial fluid of RA patients with a more pronounced production of Perforin-1 in PTPN22 risk allele carriers. Altogether, we propose a novel mechanism of action of PTPN22 risk allele through the generation of cytotoxic CD4+ T cells and identify EOMES+ CD4+ T cells as a relevant T-cell subset in RA pathogenesis.

Epigenetic-mediated immune suppression of positive co-stimulatory molecules in chemoresistant ovarian cancer cells.

The immunological response against cancer is a critical balance between immune-activating and immune-suppressing mechanisms. Ovarian cancer creates a suppressive microenvironment to escape immune elimination; however, the molecular mechanisms are poorly understood, and it is unclear whether chemotherapeutic drugs exert an immunoreactive or immunosuppressive effect on the tumor microenvironment. 4-1BB ligand (4-1BBL/CD157) and OX-40 ligand (OX-40L/CD252) are important regulators of effector cytotoxic T-cells activity. This study demonstrates that expression of positive co-stimulatory molecules, OX-40L and 4-1BBL, is suppressed while expression of immunosuppressive molecule programmed death ligand-1 (PD-L1/CD274) is enhanced in chemoresistant cells compared to parental chemosensitive ovarian cancer cells. Here, the molecular mechanisms of silencing of OX-40L and 4-1BBL expression were investigated in chemoresistant A2780-AD ovarian cancer cells. The suppression of OX-40L and 4-1BBL are due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to gene silencing during cancer progression. We identify important epigenetic regulators, histone deacetylase 1/3 (HDAC1/HDAC3) and DNA methyltransferase 1 (DNMT1), that exhibit aberrant association with OX-40L and 4-1BBL promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increase OX-40L and 4-1BBL expression in chemoresistant cells. This study suggests that loss of histone acetylation and accumulation of DNA methylation correlates with suppressed expression of OX-40L and 4-1BBL in chemoresistant ovarian cancer cells. This study marks the first report of the regulation of these two molecules by histone deacetylation and DNA methylation in chemoresistant ovarian cancer cells.

Vaccination with Irradiated Autologous Tumor Cells Mixed with Irradiated GM-K562 Cells Stimulates Antitumor Immunity and T Lymphocyte Activation in Patients with Recurrent Malignant Glioma.

PURPOSE: Recurrent malignant glioma carries a dismal prognosis, and novel therapies are needed. We examined the feasibility and safety of vaccination with irradiated autologous glioma cells mixed with irradiated GM-K562 cells in patients undergoing craniotomy for recurrent malignant glioma. EXPERIMENTAL DESIGN: We initiated a phase I study examining the safety of 2 doses of GM-K562 cells mixed with autologous cells. Primary endpoints were feasibility and safety. Feasibility was defined as the ability for 60% of enrolled subjects to initiate vaccination. Dose-limiting toxicity was assessed via a 3+3 dose-escalation format, examining irradiated tumor cells mixed with 5 × 10(6) GM-K562 cells or 1 × 10(7) GM-K562 cells. Eligibility required a priori indication for resection of a recurrent high-grade glioma. We measured biological activity by measuring delayed type hypersensitivity (DTH) responses, humoral immunity against tumor-associated antigens, and T-lymphocyte activation. RESULTS: Eleven patients were enrolled. Sufficient numbers of autologous tumor cells were harvested in 10 patients, all of whom went on to receive vaccine. There were no dose-limiting toxicities. Vaccination strengthened DTH responses to irradiated autologous tumor cells in most patients, and vigorous humoral responses to tumor-associated angiogenic cytokines were seen as well. T-lymphocyte activation was seen with significantly increased expression of CTLA-4, PD-1, 4-1BB, and OX40 by CD4(+) cells and PD-1 and 4-1BB by CD8(+) cells. Activation was coupled with vaccine-associated increase in the frequency of regulatory CD4(+) T lymphocytes. CONCLUSIONS: Vaccination with irradiated autologous tumor cells mixed with GM-K562 cells is feasible, well tolerated, and active in patients with recurrent malignant glioma. Clin Cancer Res; 22(12); 2885-96. ©2016 AACR.

Expansion of NK cells from PBMCs using immobilized 4-1BBL and interleukin-21.

Adoptive transfer of NK cells has been widely applied clinically for cancer immunotherapy. However, the difficulties to obtain a large number of activated NK cells impede the successful application of such therapy. In the present study, we implemented a novel method involving the use of immobilized human 4-1BBL and interleukin-21 to amplify NK cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors. Following stimulation for 21 days, we achieved considerable expansion of NK cells with high purity and strong cytotoxicity. This is the first time solid phase cytokines were used to augment NK cells, and this method has the advantage of no need to introduce feeder cells, without prior purification of NK cells and it effectively stimulated and expanded NK cells. The strategy of cell proliferation and activation could lead to a safer and more effective application of NK cells clinically.

4-1BB ligand signaling to T cells limits T cell activation.

4-1BB ligand (4-1BBL) and its receptor, 4-1BB, are both induced on T cells after activation, but little is known about the role of 4-1BBL. In this study we show that 4-1BBL can transmit signals that limit T cell effector activity under tolerogenic conditions. Cross-linking 4-1BBL inhibited IL-2 production in vitro, primarily with suboptimal TCR stimulation. Furthermore, naive 4-1BBL-deficient OT-II transgenic T cells displayed a greater conversion to effector T cells in vivo when responding to soluble OVA peptide in wild-type hosts, whereas development of Foxp3(+) regulatory T cells was not altered. A greater number of effector T cells also differentiated from naive wild-type OT-II T cells when transferred into 4-1BB-deficient hosts, suggesting that APC-derived 4-1BB is likely to trigger 4-1BBL. Indeed, effector T cells that could not express 4-1BBL accumulated in larger numbers in vitro when stimulated with 4-1BB-expressing mesenteric lymph node dendritic cells. 4-1BBL was expressed on T cells when Ag presentation was limiting, and 4-1BBL was aberrantly expressed at very high levels on T cells that could not express 4-1BB. Trans-ligation, Ab capture, and endocytosis experiments additionally showed that T cell-intrinsic 4-1BB regulated internalization of membrane 4-1BBL, implying that the strong induction of 4-1BB on T cells may counteract the suppressive function of 4-1BBL by limiting its availability. These data suggest that 4-1BBL expressed on T cells can restrain effector T cell development, creating a more favorable regulatory T cell to effector cell balance under tolerogenic conditions, and this may be particularly active in mucosal barrier tissues where 4-1BB-expressing regulatory dendritic cells present Ag.

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA, combined with soluble IL-21.

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56(bright)CD16(+) subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer.

Use of CD137 ligand expression in the detection of small B-cell lymphomas involving the bone marrow.

Staging for small B-cell lymphomas is important for prognostic and therapeutic decision making; however, the detection of lymphoid infiltrates in the bone marrow is often hampered by the lack of specific diagnostic markers. We recently described the hematopoietic tissue distribution patterns of CD137 and CD137 ligand (CD137L), which have shown promise as immunotherapeutic targets. CD137 expression was primarily confined to cells in the microenvironment, whereas CD137L was expressed in neoplastic cells in most B-cell lymphomas. Here we evaluate the use of CD137L in the detection of small B-cell lymphomas involving the bone marrow. To test the potential efficacy of CD137L in detecting bone marrow lymphoid infiltrates, 166 small B-cell lymphomas were evaluated by immunohistochemistry and double-immunofluorescence labeling on formalin-fixed, paraffin-embedded bone marrow core biopsies. CD137L was highly expressed in bone marrows involved by small B-cell lymphomas and included hairy cell leukemia, mantle cell lymphoma, follicular lymphoma, B-lymphoblastic leukemia, and chronic lymphocytic leukemia. In addition, a small subset of marginal zone lymphoma and most of lymphoplasmacytic lymphoma showed staining. Normal bone marrow cells including myeloid, erythroid and megakaryocytic precursors, and reactive lymphoid aggregates lacked staining. Our findings show that immunohistochemistry for CD137L is capable of reliably distinguishing small B-cell lymphomas from reactive lymphoid aggregates. These data also suggest that CD137L is useful in providing staging information for clinical diagnosis and is likely to furnish a potential target for minimal residual disease assessment as well as immunotherapy in patients with stage 4 disease.

Nicotine up-regulated 4-1BBL expression by activating Mek-PI3K pathway augments the efficacy of bone marrow-derived dendritic cell vaccination.

PURPOSE: To explore the role of 4-1BBL in nicotine-treated immature dendritic cells (imDCs) mediated anti-tumor effects. METHODS: Bone marrow-derived imDCs were stimulated with nicotine and 4-1BBL expression was determinated by flow cytometry, Western blot and RT-PCR respectively. Then, the roles of 4-1BBL in nicotine-augmented DCs-dependent T cell proliferation, CTL priming and anti-tumor effects were investigated by BrdU cell proliferation assay, enzyme-linked immunospot assay and in vivo preventive effect on tumor development, respectively. Finally, using relative kinase inhibitors, the mechanism of 4-1BBL up-regulation by nicotine stimulation and the roles of Mek-PI3K signal pathways in nicotine-augmented DCs-dependent T cell proliferation were explored by Western blot and BrdU cell proliferation assay, respectively. RESULTS: Firstly, nicotine could up-regulate 4-1BBL expression in both protein and mRNA levels. Secondly, the effects of nicotine-augmented DCs-dependent T-cell proliferation, CTL priming and anti-tumor effects could be significantly abolished by blocking CD80, CD86 and 4-1BBL activity, respectively. Thirdly, the combined blockages of CD80/CD86, CD80/4-1BBL, CD86/4-1BBL or CD80/CD86/4-1BBL signals could decrease 53.2 %, 29.6 %, 27.9 % and 54.5 % nicotine-enhanced T cell proliferation, respectively. Importantly, nicotine-induced 4-1BBL up-regulation could be decreased by the usage of Mek-PI3K pathway kinase inhibitors. The pre-treatment of Mek-p38-PI3K kinase inhibitors could obviously abolish nicotine-augmented DCs-dependent T cell proliferation. CONCLUSIONS: CD80/CD86 and 4-1BBL are critical for nicotine augmented DCs-mediated anti-tumor effects. 4-1BBL and CD80/CD86 could be considered as potential candidates for preventive and therapeutic tumor vaccination.

A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg.

Natural regulatory T cells (nTreg) play a central role in the induction and maintenance of immunological tolerance. Experimental transplant models and recent clinical trials demonstrate that nTreg can control alloreactivity. To upgrade Treg-based cell therapies to a selective suppression of undesired immune reactions, only the transfer of Ag-specific nTreg represents the appropriate therapeutic option. However, Ag-specific nTreg are present at extremely low frequencies in the periphery, and so far appropriate surface markers for their precise identification are missing. In this study, we demonstrate that activated nTreg and activated conventional T cells differ in their 4-1BB and CD40 ligand (CD40L) expression signatures, allowing a clear dissection from each other. Based on the expression of 4-1BB and absence of CD40L expression, human alloantigen-reactive Foxp3(+) nTreg can be directly isolated from MLR cultures with high purity. Alloantigen-reactive 4-1BB(+)CD40L(-) nTreg were characterized by a completely demethylated Treg-specific demethylated region and showed alloantigen-specific suppressive properties superior to polyclonal Treg. Importantly, isolated 4-1BB(+)CD40L(-) nTreg maintain the nTreg phenotype and alloantigen-reactivity after in vitro expansion. Our results offer the possibility to simultaneously analyze Ag-specific nTreg and conventional T cells, and to establish cellular therapies with Ag-specific nTreg aiming at a specific inhibition of unwanted immunity.

Long-term proliferation of functional human NK cells, with conversion of CD56(dim) NK cells to a CD56 (bright) phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12.

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation. The expanded NK cells are predominantly CD56(bright), and we show that isolated CD56(dim)CD16(+) NK cells can switch to a CD56(bright)CD16(-) phenotype and proliferate in response to 4-1BBL + IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required 'help' from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL + IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56(bright) and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.

Natural killer cell engineering for cellular therapy of cancer.

Natural killer (NK) cells can kill transformed cells and represent a promising tool for the treatment of cancer. Their function is governed by a balance of stimulatory and inhibitory signals triggered by surface receptors. Advances in NK cell therapy require the development of dependable methods for obtaining an adequate number of effector cells; additional activation or genetic modification may further increase their anticancer capacity. A method for NK cell expansion used in our laboratory relies on a genetically modified form of the K562 myeloid leukemia cell line, engineered to express a membrane-bound form of interleukin-15 and the ligand for the costimulatory molecule 4-1BB (CD137). Expanded NK cells can be transduced with genes encoding chimeric antigen receptors that stimulate tumor cell-specific cytotoxicity. These methods for NK cell expansion and genetic modification have been adapted to large-scale, clinical-grade, Current Good Manufacturing Practice conditions and support two active clinical trials. Summarized are current efforts for NK cell immunotherapy for cancer and future perspectives.

Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.

A large number of natural killer (NK) cells with high function are expected to generate especially in tumor adoptive immunotherapy. Here K562 cells were genetically modified to co-express major histocompatibility complex class I chain-related protein A (MICA), 4-1BB ligand, and IL-15, called K562-MICA-4-1BBL-IL-15. The modified K562 cells not only promoted activation, proliferation, and survival of NK cells, but also enhanced NK cell cytotoxicity. In long-term culture tests, K562-MICA-4-1BBL-IL-15 cells stimulated NK cell to expand mean 550 folds in 24-day culture and to cover from 14.8% of total peripheral blood monoclonal lymphocytes on day 1 to 86.7% on day 24. Prevalent NK cells after expansion enhanced the ability of killing targets and producing interferon gamma (IFN-γ), and kept high expression of activating receptors. The results indicated that K562-MICA-4-1BBL-IL-15 cells would be developed for expansion of NK cells ex vivo and may have important implications for clinical immunotherapy.

Doxorubicin enhances CD4(+) T-cell immune responses by inducing expression of CD40 ligand and 4-1BB.

Chemotherapy agents have adverse immunotherapeutic effects secondary to inhibition of hematopoietic stem cell proliferation, particularly in committed lymphoblast. Certain anti-cancer drugs have immuno-modulatory properties, although mechanisms are still not fully understood. In the current study, we explored the effects of doxorubicin on peripheral blood CD4(+) and CD8(+) T-cell responses pre- and post-stimulation. Doxorubicin treatment alone had no effects on peripheral blood T lymphocytes and regulatory T-cells in vivo and in vitro. However, CD4(+) T-cells were resistant to doxorubicin and demonstrated more robust proliferation than CD8(+) T-cells after doxorubicin pre-treatment. CD40 ligand and 4-1BB expression on the surface of CD4(+) T-cells were increased after TCR-ligation activation; however, expression on CD8(+) T-cells was not increased. Dendritic cells cultured in the presence of activated CD4(+) T-cells pre-treated with doxorubicin had greater survival rates than those cultured with activated CD8(+) T-cells. Doxorubicin pre-treatment followed by anti-CD3epsilon+anti-4-1BB activation led to proliferation of CD4(+) T-cells and no proliferative effects on CD8(+) T-cells. Our results collectively suggest that doxorubicin pre-treatment in cancer patients may be a more effective way to enhance anti-cancer immune responses by increased antigen-specific CD4(+) Th1 immune responses.

Decreased 4-1BB expression on HIV-specific CD4+ T cells is associated with sustained viral replication and reduced IL-2 production.

CD4+ T cell dysfunction in subjects with chronic HIV infection is in part due to an imbalance of costimulatory and coinhibitory receptors. We report that virus-specific CD4+ T cells expressing 4-1BB (CD137) or OX40 (CD134) produced more IL-2 than cells lacking these costimulatory receptors (P<0.05) and that 4-1BB was expressed at a lower level on HIV- than CMV-specific IFN-gamma and IL-2 producing CD4+ T cells (P<0.0001 and P<0.01, respectively). Suppression of viral replication with antiretroviral therapy was associated with increased 4-1BB expression on HIV- and CMV-specific IL-2 producing CD4+ T cells (P<0.05 and P<0.01, respectively) and the percentage of IL-2 producing HIV-specific CD4+ T cells that expressed 4-1BB was inversely correlated with HIV plasma viral load (r=-0.75, P=0.007). These findings indicate that the loss of 4-1BB on HIV-specific CD4+ T cells is associated with viral replication and that it may contribute to reduced IL-2 production observed during chronic infection.

Endogenous 4-1BB ligand plays a critical role in protection from influenza-induced disease.

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

4-1BB (CD137) ligand enhanced anti-tumor immune response against mouse forestomach carcinoma in vivo.

Cancer occurrence and development has been demonstrated to be associated with escape from immune surveillance, and low costimulatory molecules expression has been considered as one of the important reasons for cancer evading the immune system. 4-1BB (CD137) is a costimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) expressed on antigen presenting cells (APCs) has been shown to amplify T-cell mediated immunity. We therefore examined whether murine cancer cells expressing 4-1BBL could produce anti-tumor effects in inoculated mice. Mouse forestomach carcinoma (MFC) cells were transfected with 4-1BBL gene (MFC/4-1BBL). The proliferation of the transduced cells in vitro was not different from that of parental cells. However, MFC/4-1BBL cells developed small tumors and induced higher cytotoxicity of tumor infiltration lymphocyte (TIL). Production of cytokines (IFN-gamma, TNF-alpha and IL-2) in serum and cytotoxic T lymphocyte (CTL) activity of splenocytes from mice immunized with mitomycin C (MMC)-treated MFC/4-1BBL cells were significantly higher than that from mice immunized with MMC-treated parental MFC and MFC/pMKITneo cells. These results suggest that modification of cancer cells with 4-1BBL gene can produce anti-tumor immune responses.

Preferential T cell expansion by artificial antigen presenting cells expressing 4-1BBL alone or in combination with CD80 or CD86.

BACKGROUND: Varieties of artificial antigen presenting cells (aAPCs) with different efficiencies have been introduced to expand whole T cell population or antigen specific ones for the purpose of T cell therapy. From antibody coated beads to gene modified dendritic cells each has some advantages and disadvantages. However, no one can ignore the importance and the necessity of costimulation interaction during T cell activation. OBJECTIVE: This study was designed to compare the effectiveness of CD80/CD86 and 4-1BBL, two major costimulatory families, in costimulation of autologous T cell responses. METHODS: We used recombinant non-replicative adenoviral vectors and transferred genes of these ligands to autologous blood monocytes and skin fibroblasts to create aAPCs system. T cell response to anti-CD3 pan stimulation and some viral peptide Ags, in co-culture with gene modified monocytes and fibroblasts were studied using CFSE and HLA tetramers, respectively. RESULTS: Over-expression of ligands was able to expand the T cell population significantly higher than normal cells with no interference with antigen stimulation. Presence of 4-1BBL alone or in combination with B7 members enhanced T cell expansion and promoted more Ag-specific cells to accumulate in these culture systems. CONCLUSION: Considering the inhibitory proportion of B7 costimulation route, 4-1BBL, as an alternative signaling pathway, in combination with B7 will promote T cell proliferation and expansion.

Induction of a VLA-2 (CD49b)-expressing effector T cell population by a cell-based neuroblastoma vaccine expressing CD137L.

In malignancies where no universally expressed dominant Ag exists, the use of tumor cell-based vaccines has been proposed. We have modified a mouse neuroblastoma cell line to express either CD80 (B7.1), CD137L (4-1BBL), or both receptors on the tumor cell surface. Vaccines expressing both induce a strong T cell response that is unique in that among responding CD8 T cells, a T effector memory cell (T(EM)) response arises in which a large number of the T(EM) express the alpha-chain of VLA-2, CD49b. We demonstrate using both in vitro and in vivo assays that the CD49b(+) CD8 T cell population is a far more potent antitumor effector cell population than nonfractionated CD8 or CD49b(-) CD8 T cells and that CD49b on vaccine-induced CD8 T cells mediates invasion of a collagen matrix. In in vivo rechallenge studies, CD49b(+) T cells no longer expanded, indicating that CD49b T(EM) expansion is restricted to the initial response to vaccine. To demonstrate a mechanistic link between the expression of costimulatory molecules on the vaccine and CD49b on responding T cells, we stimulated naive T cells in vitro with artificial APC expressing different combinations of anti-CD3, anti-CD28, and CD137L. Although some mRNA encoding CD49b was induced by combining anti-CD3 with anti-CD28 or CD137L, the highest level was induced when all three signals were present. This indicates that CD49b expression results from additive costimulation and that the level of CD49b message serves as an indicator of the effectiveness of T cell activation by a cell-based vaccine.