Expression Of 4-1BBL Research Articles

Abstract 5595: Antitumor immune responses to Nitroreductase/CB1954 and 4-1BBL gene therapy

Abstract Gene-directed enzyme prodrug therapy using the non-mammalian nitroreductase enzyme (NTR) and CB1954 prodrug is a promising approach for local management of cancer through targeted chemotherapy. Whilst there is some evidence that tumor cell killing via NTR/CB1954 may also induce a systemic, immune bystander effect, this needs further investigation. To address this, a transgenic adenocarcinoma of the mouse prostate tumor cell line stably expressing cytoplasmic ovalbumin (TRAMPOva) was transduced with retroviruses encoding either the therapeutic NTR or 4-1BBL gene. The generated sub-clones were used to establish subcutaneous tumor in syngeneic mice. Adoptive transfer of Ova-specific transgenic (OT-1) T cells facilitates tracking of the specific anti-tumor immune response to the gene therapy in vivo. By examining the effect of NTR/CB1954 mediated cytotoxicity on the generation of effector memory T cells, we find that treatment of Ova+NTR+ tumor bearing mice with prodrug induced significant tumor regression. This was associated with enhanced CD8+ T cell maturation and cytolytic activity. However, adoptive transfer of 1×105 of these Ova-specific memory cells did not provide prophylactic protection to immunodeficient mice against TRAMPOva tumor challenge. Therefore, we attempted to enhance the immunogenicity of tumors by delivering the 4-1BBL costimulatory ligand gene to tumor cells. We demonstrate that immunocompetent mice receiving TRAMPOva+4-1BBL cells resisted tumor formation in a 120 day follow-up period, while those receiving TRAMPOva cells developed progressive tumors at earlier time points. Tumor rejection was accompanied by generation of higher frequencies of Ova-specific CD8+ memory T cells that showed effector cytolytic function following challenge with splenocytes loaded with Ova peptide in vivo. Overall, our results show that either NTR/CB1954-mediated cell death, or tumor expression of 4-1BBL can induce a tumor-specific CD8+ T cell response. Combining these approaches may offer a strategy to provide long-lasting anti-tumor immunity in vivo. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5595.

The expression of 4-1BBL in different gastric cancer cell lines

Objective To study the expression of 4-1BBL and its biological function in different human gastric cancer cell lines. Methods The expression of 4-1BBL in different human gastric cancer cell lines was detected by using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The killing activity of lymphocytes against gastric cancer cell lines was measured by MTT, and interleukin (IL)-2, IL-10 and tumor necrosis factor (TNF)-ct were determined in the supernatant by en-zyme-linked immunosorbent assay (ELISA). Results The RT-PCR revealed that the 4-1BBL mRNA was expressed in MKN-28 (46. 63% ), SGC-7901 (37. 13% ), BGC-823(20. 43% ) and MGC-803 (14. 14% ) in the sequence from high to low levels. The 4-1BBL protein was expressed in MKN-28, SC, C-7901, BGC-823 and MGC-803 in the sequence from high to low levels. MTF analysis indicated that the killing activity was found from high to low in MKN-28, SGC-7901, BGC-823, MGC-803. ELISA analysis showed that IL-2 and TNF-α were found in MKN-28, SGC-7901, BGC-823 and MGC-803, and IL-10 was found in MGC-803, BGC-823, SGC-7901 and MKN-28 from high to low. Conclusion The four different gastric cancer cell lines all express 4-1BBL at the mRNA and protein level. The expression level of 4-1 BBL is related to dif-ferentiation of gastric cancer cell lines. The killing activity of lymphocytes against gastric cancer cell lines is increases with the increased 4-1BBL expression. Moreover, 4-1BBL can regulate tumor immunity by pro-moting the production of TNF-α, IL-2 and IL-10. Key words: Gastric carcinoma; Tumor immunity; 4-1BBL

Retrovirally expressed 4-1BBL dramatically increases proliferation and in vivo persistence of adoptively transferred CD19-targeted T cells, promoting complete tumor elimination. (131.16)

Abstract Human T cells transduced with a chimeric antigen receptor (CAR) composed of an antibody-derived scFv fragment and the CD3ζ chain can recognize and kill tumor cells on binding to the targeted membrane antigen. The co-transduction of a CAR along with CD80 and 4-1BBL induces auto-costimulation enabling high in vivo T cell expansion and tumor rejection in a prostate tumor model. Here we investigated this concept in T cells harboring either a first generation anti-CD19 CAR (19z1) or a second generation CAR (1928z), which additionally comprises the CD28 cytoplasmic domain. We compared the proliferation and anti-tumor activity of 19z1+, 19z1-4-1BBL+, 19z1-CD80+4-1BBL+, 1928z+ and 1928z-4-1BBL+ transduced T cells in a pro B cell leukemia model (NALM-6). Seven days after IV injection into NALM-6 bearing NOD/SCID/IL2Rγnull mice, 1928z-4-1BBL+ CD8 and CD4 T cells accumulated in the bone marrow and the spleen 100 times more than 19z1+ T cells and 10 times more than 1928z+ T cells, corroborating in vitro proliferative responses to repeated antigen exposure. An increased proportion of central memory CD8 T cells was observed in all groups treated with 4-1BBL-expressing cells. Within 15 days, in vivo bioluminescence imaging showed total tumor regression in the 1928z-4-1BBL+ T cell-treated group and partial regression in the other groups. Thus, constitutive 4-1BBL expression drives high proliferation of anti-tumor CD8 T cells, promoting central memory T cell persistence and tumor eradication.

The Change of Immunoactivity of Dendritic Cells Induced by Mouse 4-1BBL Recombinant Adenovirus

PurposeThe purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells.Materials and MethodsMouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4-1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E. coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary moleculs (CD80 and CD86) on DCs were analyzed by flow cytometry.ResultsThe levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs.ConclusionThe results indicated the recombinant mouse 4-1BBL can effectively activate DCs.

Adjuvantive effects of anti-4-1BB agonist Ab and 4-1BBL DNA for a HIV-1 Gag DNA vaccine: Different effects on cellular and humoral immunity

Plasmid DNA immunizations induce low levels but a broad spectrum of cellular and humoral immune responses. Here, we investigate the potential of co-stimulation through 4-1BB as an adjuvant for a HIV-1 DNA vaccine in mice. We designed plasmid DNAs expressing either the membrane bound or soluble form of 4-1BBL, and compared with the agonistic anti-4-1BB Ab for their ability to adjuvant the Gag DNA vaccine. Both, anti-4-1BB agonistic Ab as well as 4-1BBL DNA enhanced the Gag-specific cellular immune responses. However, in complete contrast to the agonistic Ab that suppressed humoral immunity to Gag, 4-1BBL DNA adjuvanted vaccines enhanced Gag-specific IgG responses. Importantly, the expression of Gag and 4-1BBL from the same plasmid was critical for the adjuvant activity. Collectively, our data suggest that for a HIV-1 vaccine where both antigen-specific cellular and humoral immunity are desirable, 4-1BBL expressed by a DNA vaccine is a superior adjuvant than anti-4-1BB agonistic Ab.

Peripheral NK Cell Activation and Expansion by Artificial Antigen Presenting Cells, 2D11 (41.25)

Abstract Natural killer (NK) cells play an important role in early immune defenses against both nonself cells and autologous cells infected with viruses, bacteria, or parasites or in malignant transformation. NK cells mediate direct cytotoxic attack without prior sensitization on their targets through activating receptors, NKG2D, NCR, TRAIL, and inhibitory receptors ("absence of self"), and/or produce cytokines and chemokines that contribute to cytotoxicity and immunoregulation. A number of studies have reported that activation/expansion enhances NK cytotoxicity, but optimal methods for activating and expanding NK cells have not been well defined. Here we demonstrate that artificial APCs (2D11), previously reported and used to expand antigen-specific T cells, also efficiently expand NK cells via NKG2D and 4-1BB signaling mediated by MICA/B and 4-1BBL expression respectively on 2D11 aAPCs. In the presence of exogenous IL15, which is trans-presented by IL-15Rα on 2D11, robust expansion of highly lytic activated NK cells occurs. Compared to resting NK cells, IL15/2D11-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and exerted enhanced cytotoxicity against K562, Ewing's tumor cell lines, including autologous tumors, ALL, as well as NK-resistant Daudi cells. In previous studies, expression of KIR ligands on tumor cells has been put forth as a mechanism of tumor immune escape from NK cell mediated killing. Our preliminary results demonstrate that expression of KIR ligands on tumor targets no longer inhibits killing by NK cells activated with 2D11. Ongoing studies are underway to test the hypothesis that NK cells activated by IL15/2D11 will prove more effective therapeutics due to breaking up of KIR-mediated inhibition of tumor cell killings.

4-1BBL expressed by eukaryotic cells activates immune cells and suppresses the progression of murine tumor

The interaction by co-stimulatory molecules 4-1BB and 4-1BB ligand (4-1BBL) plays an important role in the activation, proliferation and differentiation of T lymphocytes. The function of 4-1BB/4-1BBL expressed by the immune cells has been the focus for many tumor immunotherapy efforts. In this study, 4-1BBL was expressed in non-immune cells and non-tumor cells, and the role of 4-1BBL in lymphocyte activation and tumor suppression was investigated. The plasmid p4-1BBL containing the full length of mouse 4-1BBL cDNA sequence was constructed, and the plasmid was transfected into baby hamster kidney (BHK) cells and murine muscle cells by means of lipofectin-mediated or naked plasmid DNA injection into the muscle directly. The study demonstrated that the molecule 4-1BBL expressed by BHK cells in vitro could enhance the proliferation and cytotoxicity of lymphocytes, and it could increase the expression level of IL-2 and IFN-γ. The treatment with plasmid p4-1BBL in vivo revealed that the number of CD8+ Tcells in the peri-tumoral tissue increased markedly, and the growth rate of the tumor was significantly lower than that of control group. These findings suggest that expression of 4-1BBL by normal cells in the tumor microenvironment can enhance the proliferation and other functions of T lymphocytes. This therapeutic method may provide a promising approach for tumor immunotherapy.

Recombinant attenuated Salmonella harboring 4-1BB ligand gene enhances cellular immunity

Objective To transfect antigen presenting cells (APCs) with 4-1BB ligand DNA by attenuated Salmonella enterica serovar Typhimurium in vivo, and to observe the effects of ectogenous 4-1BBL on the immune functions of infected rats. Methods Attenuated Salmonella typhimurium (vaccine strain) carrying plasmids pIRES2-EGFP-4-1BBL was constructed and used to infect HepG2 hepatoma cells. The expression of reporter gene, green fluorescent protein (GFP) and rat 4-1BBL in the transfected cells was detected by double-immunofluorescence staining. Rats were fed with the recombinant bacteria intragastrically on three occasions in 2 weeks, and were then sacrificed. The transcription and expression of GFP and 4-1BBL genes in splenocytes were measured by RT-PCR and flow cytometry. The phenotypes of T cells in peripheral blood and splenocytes were determined by flow cytometry. The content of IFN-γ in the cultural supernatant of splenocytes stimulated by PHA was measured by ELISA. Results The recombinant bacteria harboring 4-1BBL had the same invasive abilities as the original bacteria, and it was able to deliver exogenous genes into HepG2 cells, where the GFP and 4-1BBL were successfully expressed. There were significant upregulations of CD3 +CD8 + T cells ( P = 0.018) and CD3 +CD25 + T cells ( P = 0.019) in the peripheral blood cells as well as CD3 +CD8 + T cells ( P = 0.022), and CD3 +CD25 + T cells ( P = 0.008) in splenocytes of the infected rats. The rats had more 4-1BBL expression detected in the spleen. IFN-γ released by PHA-stimulated splenocytes increased significantly by the recombinant bacteria as compared with controls ( P = 0.002). Conclusion Salmonella serovar Typhimurium containing 4-1BBL can transfect target genes into antigen presenting cells in vivo, and the expression of exogenous 4-1BBL enhances cellular immunity markedly.

Expression of 4-1BB and 4-1BBL in thymocytes during thymus regeneration

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a major costimulatory receptor that is rapidly expressed on the surface of CD4(+) and CD8(+) T cells after antigen- or mitogen-induced activation. The interaction of 4-1BB with 4-1BBL regulates immunity and promotes the survival and expansion of activated T cells. In this study, the expression of 4-1BB and 4-1BBL was examined during regeneration of the murine thymus following acute cyclophosphamide- induced involution. Four-color flow cytometry showed that 4-1BB and 4-1BBL were present in the normal thymus and were preferentially expressed in the regenerating thymus, mainly in CD4(+)CD8(+) double-positive (DP) thymocytes. Furthermore, the CD4(lo)CD8(lo), CD4(+)CD8(lo) and CD4(lo)CD8(+) thymocyte subsets, representing stages of thymocyte differentiation intermediate between DP and single-positive (SP) thymocytes, also expressed 4-1BB and 4-1BBL during thymus regeneration but to a lesser degree. Interestingly, the 4-1BB and 4-1BBL positive cells among the CD4(+)CD8(+) DP thymocytes present during thymus regeneration were TCR(hi) and CD69(+) unlike the corresponding controls. Moreover, the 4-1BB and 4-1BBL positive cells among the intermediate subsets present during thymus regeneration also exhibited TCR(hi/int+) and CD69(+/int) phenotypes, indicating that 4-1BB and 4-1BBL are predominantly expressed by the positively selected population of the CD4(+)CD8(+) DP and the intermediate thymocytes during thymus regeneration. RT-PCR and Western blot analyses confirmed the presence and elevated levels of 4-1BB and 4-1BBL mRNA and protein in thymocytes during thymus regeneration. We also found that the interaction of 4-1BB with 4-1BBL promoted thymocyte adhesion to thymic epithelial cells. Our results suggest that 4-1BB and 4-1BBL participate in T lymphopoiesis associated with positive selection during recovery from acute thymic involution.

The role of soluble and cell-surface expressed 4-1BB ligand in patients with malignant hemopoietic disorders

The TNFR family member 4-1BB and its ligand 4-1BBL are involved in the costimulation of T-cells and tumor-derived soluble (s)4-1BBL may influence the interaction of malignant cells with the immune system. Here, we report that cell-surface-expressed (c)4-1BBL can be expressed on mononuclear blood cells from patients with acute myeloid leukemia (AML) (n = 35), myelodysplasia (n = 5) or non-Hodgkin lymphoma (n = 11) and can be coexpressed on varying proportions of lymphoid or myeloid malignant cells and on dendritic cells differentiated from AML-blasts. Direct correlations between c- and s4-1BBL were not found in the investigated cases. Up to now expression of 4-1BBL has not been described on primary myeloid malignant cells, but only on malignant cells of lymphoid or solid tumor origin or on tumor cell lines. With our work we further contribute to the understanding of the potential role of c/s4-1BBL in immune reactions and its influence on the interaction of tumor and immunoreactive cells.

Berkeley Mice with Sickle Cell Disease Are Not More Immunizable to Transfused HOD Red Blood Cells Than Sickle Trait Control Mice

Background: Patients with sickle cell disease have higher rates of red blood cell (RBC) alloimmunization following transfusion than any other patient population. However, it is unclear if they are inherently more immunizable, or if the high rates of RBC alloimmunization are simply due to antigenic differences between donors/recipients and heightened transfusion frequency. Utilizing a murine model, we have previously shown that rates of alloimmunization are influenced by the inflammatory status of the recipient at the time of the transfusion. Thus, we hypothesized that sickle patients may be more immunizable, due in part to the baseline inflammation associated with their disease. Herein, we present a reductionist murine sickle model of RBC alloimmunization, and investigate the hypothesis that mice with sickle cell disease are more likely to become alloimmunized following transfusion of RBCs containing a foreign antigen than sickle trait controls.Materials/Methods: Berkeley mice (with human α and βS-globin) were determined to be homozygous (sickle cell disease) or heterozygous (sickle cell trait) by cellulose acetate electrophoresis. We generated the HOD mouse, with RBC specific expression of the model humoral antigen hen egg lysozyme (HEL) fused to the model minor histocompatibility antigen ovalbumin (OVA), linked to the cell membrane by a human blood group antigen (Duffy). The inclusion of OVA in the HOD construct allows presentation of HOD peptides from H-2b MHC; thus, Berkeley mice are able to process and present the HOD antigen. 100 microliters of packed HOD RBCs were transfused IV into mice with sickle cell disease or sickle cell trait; alloimmunization to the HEL antigen was assessed by anti-HEL IgG ELISA 2 weeks following transfusion. Given that co-stimulatory molecule expression on antigen presenting cells in part determines the response of the CD4+ T cell to the antigen being presented, we examined baseline expression of B7-1, B7-2, Ox40L, CD70, 41BBL, CD30L, and CD40 on macrophages (F480 high) and dendritic cells (CD11c high) in sickle cell disease and sickle trait mice.Results: Data for a standard curve was fit to one phase exponential association, with a non-linear best fit regression analysis (R2 of 0.96). Utilizing the OD 415 value of the anti-HEL IgG ELISA, data was combined from 3 separate experiments (n=31 mice). An unpaired two-tailed t-test showed a mean of 6.25e-5, SD 5.87e-5 for sickle disease mice, and a mean of 4.14e-5, SD 2.84e-5 for sickle trait mice, p=0.217 (see figure). Baseline co-stimulatory molecule expression of all molecules examined on macrophages and dendritic cells was similar in both sickle cell disease and sickle trait mice. [Display omitted] Conclusions: Generation of the HOD mouse, containing a model humoral antigen capable of being presented by the antigen presenting cells in the Berkeley mice, allowed these studies to be performed. These data rule out the hypothesis that Berkeley mice with sickle cell disease are substantially more immunizable to a single transfusion of RBCs containing the HOD foreign antigen than are those with sickle cell trait. However, there is a large standard deviation in alloantibody response between individual mice with sickle cell disease. The varied response may be explained by differing levels of illness in individual mice; ongoing studies are investigating such a potential correlation. Clinically, many patients with sickle cell disease are transfused in times of illness (i.e. acute chest syndrome), and the possibility that illness alters alloantibody response cannot be ruled out by these studies. Furthermore, although the model was designed to be as reductionist as possible, these studies are limited by the highly heterogeneous genetic background of the Berkeley mice (including FVB/N, 129, DBA/2, C57BL/6, and Black Swiss); this diverse background likely influences response to foreign antigen and makes identifying control groups difficult. Finally, although Berkeley mice with sickle cell disease are known to have baseline inflammation associated with their disease, this does not appear to influence co-stimulatory molecule expression on their antigen presenting cells.

Activated B cells modified by electroporation of multiple mRNAs encoding immune stimulatory molecules are comparable to mature dendritic cells in inducing in vitro antigen‐specific T‐cell responses

Ex-vivo-activated B cells are an alternative source of antigen-presenting cells (APCs) and a potential replacement for dendritic cells (DCs) in immunotherapy. However, the ability of ex-vivo-activated B cells to function as potent APCs has been a concern, especially when compared to DCs. Our study investigated whether modification of activated B cells with immune stimulatory molecules could enhance the ability of activated B cells to stimulate T cells. We show that murine splenic B cells, activated with a combination of Toll-like receptor agonist and agonistic anti-CD40, stimulated antigen-specific CD8+ T cells more efficiently than cells activated with Toll-like receptor agonist or anti-CD40 alone, probably by down-regulation of the immune regulatory cytokine interleukin-10 (IL-10). However, the activated B cells were still poor T-cell stimulators compared to mature DCs. Therefore, we modified the activated B cells by simultaneous electroporation of multiple messenger RNAs encoding costimulatory molecules (OX40L and 4-1BBL), cytokines (IL-12p35 and IL-12p40) and antigen. We found that de novo expression or overexpression of OX40L, 4-1BBL and IL-12p70 on activated B cells synergistically enhanced proliferation as well as IL-2 and interferon-gamma production by CD8+ T cells. Furthermore, the RNA-modified activated B cells induced antigen-specific cytotoxic T lymphocyte responses as efficiently as mature DCs in vitro. Unexpectedly, modified activated B cells were inferior to mature DCs at in vivo induction of CD8+ T-cell responses. In summary, activated B cells modified to express immune stimulatory molecules are a potent alternative to DCs in immunotherapy.

Suppression of neointimal hyperplasia after vascular injury by blocking 4-1BB/4-1BB ligand pathway

T cell-mediated immunity is involved in the pathogenesis of atherosclerosis and arteriosclerosis. This study examined whether the 4-1BB pathway affects the development of arteriosclerosis after vascular injury. The left or right femoral arteries of adult male mice weighing 22 to 25 g were injured with a straight spring wire. The injured artery was excised 28 days later. Confocal microscopy revealed intense expression of both 4-1BB and 4-1BBL in the developing neointima and media. Similar results were obtained on immunoblotting analysis of lysates of the injured arteries. We gave mice an injection of 100 microg or 200 microg 4-1BB-fused with human immunoglobulin (Ig) every other day over the course of 5 days. As compared with untreated controls (intima/media ratio, 2.13 +/- 0.37, n=10), the intima/media ratio was smaller in mice treated with 200 microg of 4-1BBIg (1.20 +/- 0.30, n=6, p<0.05), but not in mice treated with 100 microg of 4-1BBIg (1.56 +/- 0.27, n=9). 4-1BB inhibits neointimal hyperplasia after vascular injury. Our findings suggest that 4-1BB is involved in injury-induced neointimal hyperplasia and may be an effective target for the treatment of neointimal hyperplasia.

Increased Soluble 4‐1BB Ligand (4‐1BBL) Levels in Peripheral Blood of Patients with Multiple Sclerosis

4-1BB ligand (4-1BBL; CD137L) is a member of the tumour necrosis factor superfamily expressed primarily on antigen presenting cells such as B cells, macrophages and dendritic cells. Its engagement with the receptor 4-1BB (CD137) has been shown to promote T-cell activation and regulate proliferation and survival of T cells. The role of the costimulatory molecule in multiple sclerosis (MS) remains unclear. In this study, the expression of 4-1BBL and soluble 4-1BBL (s4-1BBL) protein levels were analysed in peripheral blood of MS patients. Compared with healthy controls, MS patients had an increase in both plasma s4-1BBL protein levels and expression of 4-1BBL in CD14(+) monocytes. In contrast, myelin basic protein-reactive T-cell proliferation was not found to be inhibited by the use of an anti-4-1BBL antibody. The elevated s4-1BBL protein levels in the MS patients may function as a self-regulatory mechanism of 4-1BB/4-1BBL interaction and costimulation.

Impaired maturation and altered regulatory function of plasmacytoid dendritic cells in multiple sclerosis

Plasmacytoid dendritic cells (pDCs) represent a DC subtype that exerts divergent functions in innate and adoptive immunity including the immediate reaction to microbial factors and the induction of immunoregulatory responses. It is thought that different DC subtypes may be critically involved in the pathogenesis of multiple sclerosis (MS). In our study we assessed the phenotype, maturation and functional properties of peripheral blood pDCs from 35 clinically stable, untreated multiple sclerosis patients, 30 healthy controls and 9 patients with pneumonia, which was used as a non-specific inflammatory condition (NIC). Ex vivo expression of CD86 and 4-1BBL was significantly lower on pDCs from multiple sclerosis patients than from controls and patients with NIC (22 versus 47 versus 41% and 12 versus 35 versus 32%, respectively). When stimulated with IL-3 and CD40L, pDCs of multiple sclerosis patients showed inefficient maturation as demonstrated by significantly lower or delayed upregulation of CD86, 4-1BBL, CD40 and CD83. Additionally, in multiple sclerosis, stimulation of pDCs by unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) resulted in a significantly lower interferon (IFN) alpha secretion than in controls. In multiple sclerosis, but not in controls, pDCs failed to upregulate proliferative responses and IFN-gamma secretion of autologous peripheral blood mononuclear cells (PBMC) in a co-culture system. Moreover, depletion of pDCs in multiple sclerosis patients, but not in controls, had no effect on generation of CD4+Foxp3+ regulatory T cells. We also provide data showing that glatiramer acetate (GA) treatment partially restores phenotype and function of pDCs in multiple sclerosis patients. These findings suggest functional abnormalities of pDCs in these patients, which might be of importance in the understanding of the development of immune dysregulation in this disease.

Expression of 4-1BB and 4-1BBL in Graves' Disease

Background: 4-1BB mediated costimulatory signal is a recently identified immunotherapeutic strategy for treating autoimmune diseases without depressing the immune response. In this study, we investigated the expression of 4-1BB and 4-1BBL on the peripheral blood mononuclear cells (PBMC) and we assessed whether the serum levels of soluble (s) 4-1BB and s4-1BBL in the patients with Graves' disease (GD) and compared them with normal subjects. Methods: Expression of 4-1BB and 4-1BBL on PBMC of GD patients was determined by flow cytometry. The concentrations of s4-1BB and s4-1BBL were assessed in the sera of GD patients by performing ELISA. Results: 4-1BB was constitutively expressed on naive CD4 and CD8 T cells of the GD patients and this was increased by stimulation. 4-1BBL was also expressed on the antigen-presenting cells such as CD19 B cells, monocytes and dendritic cells in GD patients. The serum levels of s4-1BB and s4-1BBL were significantly higher in GD patients than those in controls, and these levels were significantly correlated with the serum levels of thyroid-binding inhibitory immunoglobulin and free T4. Conclusion: These results indicate that effector T cells of GD patients can be activated through the 4-1BB-mediated costimulatory signal. Elevated s4-1BB and s4-1BBL levels in the sera of GD patients may affect modulation of the clinical course in GD patients.

Expression of Costimulatory Molecules (4-1BBL and Fas) and Major Histocompatibility Class I Chain-Related A (MICA) in Aortic Tissue with Takayasu’s Arteritis

To further investigate the immunological mechanisms involved, we analyzed the expression of costimulatory molecules in aortic tissue and their counterpart molecules on infiltrating cells of patients with Takayasu’s arteritis. We also examined the expression of major histocompatibility complex (MHC) class I chain-related (MIC) A in aortic tissue, which is known to be induced by external stress, and its counterpart NKG2D receptors on infiltrating cells. Among these costimulatory molecules, strong expression of 4-1BBL and Fas was induced in the aortic tissue, and most of the infiltrating cells expressed 4-1BB and FasL, suggesting these pathways play critical roles in T-cell-mediated vascular injury. We also found that MICA was strongly induced in the aortic tissue and that at least part of the infiltrating cells expressed NKG2D receptors. Some infiltrating cells – but not vascular smooth muscle cells – seemed to have undergone apoptosis. Our findings strongly suggest that 4-1BB/4-1BBL and Fas/FasL pathways play important roles in vascular injury in Takayasu’s arteritis. We assume that γδ T cells infiltrated aortic tissue recognizing MICA, resulting in the induction of MHC antigens and costimulatory molecules, and then αβ T-cells infiltrated recognizing some auto-antigens presented by MHC antigens, leading to chronic inflammation.

Anti-CD137 antibodies in the treatment of autoimmune disease and cancer.

CD137 (4-1BB), is an inducible T-cell costimulatory receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily. It is expressed on activated T cells and activated natural killer (NK) cells, but is constitutively expressed on a population of splenic dendritic cells (DCs). The natural counter receptor for CD137 is 4-1BB ligand, a member of the TNF superfamily that is weakly expressed on naïve or resting B cells, macrophages, and DCs. Upon activation, the level of 4-1BBL expression increases on these cells. In T cells CD137-induced signals lead to the recruitment of TRAF family members and activation of several kinases, including ASK-1, MKK, MAPK3/ MAPK4, p38, and JNK/SAPK. Kinase activation is then followed by the activation and nuclear translocation of several transcription factors, including ATF-2, Jun, and NF-kappaB. CD137-mediated T-cell costimulation as measured by enhanced proliferation and cytokine production can be induced by anti-CD137 monoclonal antibodies (MAbs) or by employing immobilized 4-1BB ligand. In addition to augmenting suboptimal TCR-induced proliferation, CD137-mediated signaling protects T cells, and in particular, CD8+ T cells from activation-induced cell death (AICD). Although studies with CD137-deficient or 4-1BBL-deficient mice failed to demonstrate any loss of essential immunological function, or other noteworthy deficits, we have found that 4-1BBL-deficient mice failed to generate a strong antiviral immune response following lymphocytic choriomeningitis virus (LCMV) peptide vaccination. We further found that although compromised, the immune response to LCMV vaccination in these mice could be fully restored by injecting them with anti-CD137 MAbs at the time of vaccination. Finally, we have found that injecting normal mice with anti-CD137 MAbs had profound effects on their ability to develop immune responses to allo- and autoantigens. The results of these studies discussed in this article provide a rationale for assessing the potential use of anti-CD137 MAbs for therapeutic purposes.

Lipopolysaccharide preferentially induces 4-1BB ligand expression on human monocyte-derived dendritic cells

Dendritic cells (DCs) represent a promising tool for immunotherapy. A key feature in their action is to provide co-stimulatory signals for full activation of T cells. In view of recent studies demonstrating the critical role of 4-1BB co-stimulation in T cell response, it is of importance to optimize 4-1BB ligand (4-1BBL) expression on human monocyte-derived DCs (MDDCs), the DC source of many clinical studies. In this study, two types of MDDCs, generated in granulocyte-macrophage colony-stimulating factor and interleukin-4 (GM-CSF/IL-4-DCs) or in interferon-β and IL-3 (IFN-β/IL-3-DCs), were analyzed for 4-1BBL expression in response to several known DC activators. Immature MDDCs expressed 4-1BBLs at very low levels. Lipopolysaccharide (LPS) was the only activator that preferentially triggered 4-1BBL expression on either MDDCs, but 4-1BBL-positive cells were significantly more frequently observed on LPS-activated GM-CSF/IL-4-DCs (30.2±2.6% versus 14.3±1.2%). Combinations of multiple activating signals did not bring about enhanced 4-1BBL stimulatory capacity. In addition, plasmid DNA transfection and necrotic cell pulsing of GM-CSF/IL-4-DCs for antigen loading also resulted in 4-1BBL up-regulation. However, in all circumstances, the induced 4-1BBL levels were low in comparison with CD80 co-stimulatory molecule. Finally, by demonstrating LPS-matured GM-CSF/IL-4-DCs from sorted 4-1BBLhigh population augmented T cell expansion and survival, we propose that efforts are required to increase 4-1BBL levels on MDDCs achieved by current activation schemes.

Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen.

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-1BBL into murine DCs, and monitored the ability of these recombinant DCs to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DCs increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DCs. We also report an additive effect of 4-1BBL and receptor activator of NF-kappa B/receptor activator of NF-kappa B ligand coexpression in E7-transduced DC immunogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.