EL4 Lymphoma Research Articles

Abstract 1035: Targeting glutamine metabolism with the novel inhibitor JHU-083 inhibits tumor growth and alters the tumor immune microenvironment

Abstract Glutamine metabolism plays a critical role in promoting multiple biosynthetic pathways necessary for tumor growth. Therefore we endeavored to create a small molecule inhibitor that would block multiple pathways of glutamine metabolism as opposed to blocking one enzyme. Previous studies employing the non-specific glutamine antagonist 6-diazo-5-oxo-L-norlecuine (DON) have achieved this goal however, with much toxicity. To this end we devised a novel prodrug of DON (JHU-083) with distinct distribution and cellular partitioning properties. When dosed on an equimolar basis, JHU-083 retains the antitumor efficacy of DON but is devoid of the gut toxicity and morbidity/mortality. To this end Wild-type mice were injected subcutaneously EL4 T-cell lymphoma cells. The tumors were allowed to engraft and treatment was initiated on day 5 with either vehicle, DON or JHU-083. After 7 days of treatment there was complete tumor regression in both the DON and JHU-083 treated mice, while tumors continued to grow in the vehicle treated mice. Importantly, after 7 days of treatment the DON treated mice appeared ill with lethargy and ruffled fur and even death as a result of treatment toxicity. Alternatively, the JHU-083 treated mice remained healthy and vibrant. Based on these studies in a lymphoma model we next tested JHU-083 in 2 models of colon cancer. To this end we injected wild-type mice subcutaneously with MC38 or CT26 cells. Again, similar to the EL4 lymphoma experiments, we observed that JHU-083 resulted in complete regression of MC38 and CT26 tumors after 4 and 7 days of treatment respectively. The tumor microenvironment, which promotes immune evasion, is very much influenced by tumor metabolism. Thus, we wanted to determine the effect of inhibiting glutamine metabolism on the tumor immune microenvironment. To this end mice injected with B16 melanoma cells were treated for 3 days with DON during which time the tumors decreased in size. Interestingly, there was also a concomitant decrease in the percentage of Foxp3+ cells and an increase in the CD8:Foxp3+ T cell ratio amongst the TILS. Overall our studies demonstrate the principle of cellular selectivity based upon demand. That is, a non-selective inhibitor of glutamine metabolism can robustly inhibit tumor growth while demonstrating minimal side effects based on the differential demand of cancer cells versus normal cells. Furthermore, our studies suggest that sequencing anti-metabolic therapy with immunotherapy might prove to be a novel means of broadening and enhancing the role of immunotherapy for the treatment of cancer. Citation Format: Judson Englert, Chih-Hsien Cheng, Robert Leone, Pavel Majer, Rana Rais, Barbara Slusher, Jonathan Powell. Targeting glutamine metabolism with the novel inhibitor JHU-083 inhibits tumor growth and alters the tumor immune microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1035.

Tumour cell-derived exosomes endow mesenchymal stromal cells with tumour-promotion capabilities.

Mesenchymal stromal cells (MSCs) are a major component of the tumour microenvironment. A plethora of elegant studies focusing on tumour-derived MSCs have shown that they, unlike normal MSCs in other tissue, exhibit a strong ability to promote tumour progression. However, the mechanisms underlying the conversion of normal MSCs into tumour-associated MSCs are unknown. We report here a critical role of tumour cell-derived exosomes in endowing bone marrow-derived MSCs (BM-MSCs) with a tumour-favourable phenotype. Tumour cell-derived exosomes affected neither the growth factor production nor the immunosuppressive property of MSCs; rather, they endowed MSCs with a strong ability to promote macrophage infiltration into B16-F0 melanoma or EL-4 lymphoma. Ablation of macrophages by clodronate liposome administration reversed the tumour-promoting effect of MSCs educated by tumour cell-derived exosomes (TE-MSCs) on the tumour growth. By comparing the chemokine profile of BM-MSCs with that of TE-MSCs, we found that TE-MSCs produced a large amount of CCR2 ligands, CCL2 and CCL7, which are responsible for macrophage recruitment. CCR2-specific inhibitor was found to block the tumour-promoting effect of TE-MSCs. Thus, our investigations demonstrated that tumour cell-derived exosomes confer BM-MSCs the ability to enhance tumour growth. Therefore, we uncovered a novel mechanism underlying the conversion of normal MSCs to tumour-associated MSCs.

Leucine metabolism is a promising new approach to study the interplay between T cells and cancer cells

Abstract Cancer and T cells undergo similar metabolic reprogramming during proliferation to support increased biosynthetic and energy demands. They both rely on high rates of glycolysis while retaining mitochondrial respiration, a phenomenon known as the Warburg effect. In the tumor microenvironment, tumor cells can escape immune surveillance by competing with T cells for common metabolites and redirecting nutrients for their own advantage. To gain insight into the relationship between T cells and cancer cells, we studied the effect of leucine metabolism on T cells and their cancerous counterpart, EL-4 lymphoma cells. Our previous results showed that a loss of expression of leucine degrading enzymes (cytosolic or mitochondrial branched chain aminotransferases, BCATc and BCATm, respectively) increased intracellular leucine concentrations and stimulated glycolytic rate in activated T cells. These results correlated with activation of complex 1 of the mammalian target of rapamycin (mTORC1). Continuation of this study with EL-4 lymphoma cells, grown in leucine free media or treated with the leucine antagonist, n-acetyl-leucine amide, revealed a decrease in glycolysis, glycolytic capacity, and glycolytic reserve while mTORC1 was only moderately impacted. The oxygen consumption rate was stimulated in T cells lacking BCATc and/or BCATm but was decreased in the absence of leucine in EL-4 cells. The results suggest that T cells and their cancerous counterpart use leucine in a similar manner and that leucine is essential for the regulation of glucose and oxygen metabolism in either cell type. Future studies targeting leucine metabolism in the tumor microenvironment will aim to improve the effectiveness of T cells to fight cancer cells.

Transplantational and specific antitumor immunity in retrospective view: new models based on transgenesis of individual chains of T-cell receptor

Findings in experimental oncology in beginning of last century and subsequent achievements of genetics of tissue compatibility resulted in divergence of transplantational immunology and oncoimmunology. However, central achievements of both scientific fields are based on unified phenomenon of interaction between T-cell receptor (TCR) and histocompatibility molecules. In this review we describe the history of ideas, achievements and unique experience of the team of the Laboratory of Regulatory Mechanisms in Immunity at Scientific Research Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center for all time of existence. This experience shows that efficiency of immunological defense including immunological surveillance are critically influenced by T-cell receptor repertoire. Transgenesis of individual chains of TCR is one of possible means to manage T-cell repertoire. Functional outcomes of transgenesis may be different due to diverse extent of dependence of α- and β-chains expression on the rules of allelic exclusion. Expression of transgenic β-chains results in the expansion of TCR repertoire diversity. Expression of β-chains is under strong control by allelic exclusion, resulting in formation of repertoire bearing mainly invariant transgenic β-chain pared with different α-chains and overall narrowing of repertoire. Earlier, we cloned genes encoding α- and β-chains of TCR of CD8+ memory cells specific to histocompatibility molecule H-2Kb . After introduction them in zigotes we have obtained transgenic mouse strains, which could be used for modeling of interactions between tumor cells and immune system of recipient. Normally, B10. D2 (R101) mice reject lymphoma EL4 cells in 12–14 days after transplantation, in spite of the fact, that allogeneic difference between B10. D2 (R101) (Kd Id Db ) mice and lymphoma EL4 (H-2b) cells is only in one product of MHC, the H-2Kb molecule. Transgenics carrying β-chains of TCR displayed compromised immunity to tumor cells resulting to their long persistence, tumor progression, the loss of H-2Kb molecule and death in 2–3 months after transplantation. This model allows to see all three phases of interaction between tumor and immune system of recipient – elimination, equilibrium and escape. Against, transgenics carrying α-chain reject tumor cells much more quickly, as in secondary immune response, in 3–6 days after transplantation. This rejection was mediated by intraepithelial T lymphocytes displaying features of resident memory cells – inability to recirculation, expression of CD103 and early activation antigen CD69 and intermediate density of T-cell markers CD3 and CD8. The capability to be located in nonlymphoid tissue and quickly destroy tumor cells makes them to be the most probable candidate to perform immunological surveillance functions.

473 An IL-1a producing dermal macrophage enhances adaptive immunity to a broad group of tumors in skin

IL-1a and IL-1b are the oldest members of the growing IL-1 family of molecules. Mice deficient in IL-1b are viable and are grossly indistinguishable from WT mice. However, they are uniquely effective at rejecting malignant tumor cell lines that grow readily in syngeneic mice. Significant growth reduction of tumors formed by EL4 lymphoma, B16 melanoma, and Lewis Lung carcinoma cells implanted in the skin of IL-1b-/- were all observed, and in many cases tumors grew initially and then regressed completely. These latter mice remained completely resistant to subsequent implantation of tumor cell lines, even at very high numbers. Surprisingly, tumors grew unimpeded in IL-1a-/- and IL-1R-/- mice at a rate indistinguishable from tumors in WT mice. Additional studies demonstrated that antibody depletion of T cells from IL-1b-/- mice completely abrogated the tumor resistance, and that the tumor resistance could be partially mimicked in WT mice with a blocking antibody to IL-1b. Interestingly, the IL-1b-/- tumor resistance effect required the presence of IL-1a. Bone marrow chimera studies indicated that the tumor resistance effect required type I IL-1R to be present on T cells, but also showed that the relevant IL-1b-/- cell mediating the effect produced IL-1a, was not bone marrow derived, and was radioresistant. We speculated that the responsible cell meeting these criteria might be a dermal macrophage, and thus treated mice intradermally with chlodronate, which very effectively depletes macrophages in this setting. Injection of clodronate at day 2 after tumor implantation, and then weekly thereafter, completely abrograted the protective anti-tumor affect in IL-1b-/- mice. Thus, IL-1a, in the absence of IL-1b, appears to have strong stimulatory affect on the adapative immune system that can be harnessed for anti-tumor immunity. Combining antibodies to IL-1b with checkpoint inhibitor antibodies to enhance tumor immunity is a rational goal.

The structure-dependent toxicity, pharmacokinetics and anti-tumour activity of HPMA copolymer conjugates in the treatment of solid tumours and leukaemia

Polymer drug carriers that are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been widely used in the development and synthesis of high-molecular-weight (HMW) drug delivery systems for cancer therapy. In this study, we compared linear (Mw ~27kDa, Rh ~4nm) and non-degradable star (Mw ~250kDa, Rh ~13nm) HPMA copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazone bond. We determined the in vitro and in vivo toxicity of both conjugates and their maximum tolerated dose (MTD). We also compared their anti-tumour activity in mouse B-cell leukaemia (BCL1) and a mouse T-cell lymphoma (EL4) model. We found that MTD was higher for the linear conjugate (85mgDOX/kg) and lower for the star conjugate (22.5mgDOX/kg). An evaluation of the intestinal barrier integrity using FITC-dextran as a gut permeability tracer proved that no pathology was caused by the MTD of either conjugate. However, free DOX showed some damage to the gut barrier. The therapy of BCL1 leukaemia by both of the polymeric conjugates using the MTD or its fraction (i.e., equitoxic dosage) showed better results in the case of the star conjugate. On the other hand, treatment of EL4 lymphoma seemed to be more efficient when the linear conjugate was used. We suppose that the anti-cancer treatment of solid tumours and leukaemias requires different types of drug conjugates. We hypothesise that the most suitable HPMA copolymer-DOX conjugate for the treatment of solid tumours should have an HMW structure with increased Rh that would be stable for three to four days after the conjugate administration and then rapidly disintegrate in the short polymer chains, which are excretable from the body by glomerular filtration. On the other hand, the treatment of leukaemia requires a drug conjugate with a long circulation half-life. This would provide an active drug, whilst slowly degrading to excretable fragments.

Activation of NK and CD8+ T-Cells with a Novel IL-15 and TGF-Beta Receptor Fusion Protein Confers Anti-Tumor Immunity

The use of cytokines as agents to augment immune responses against malignancies have been dealt setbacks due to immune selection of tumors, resulting in subpopulations that elaborate tumor-derived soluble factors, such as transforming growth factor-beta (TGF-β), which suppress immune effector functions. TGF-β is overexpressed by many solid and hematological malignancies and is well known to inhibit the proliferation and anti-tumor functions of lymphomyeloid cells.In order to maximize cytokine-based immunotherapy against tumors, we have designed a novel fusion protein consisting of proinflammatory murine interleukin-15 (IL-15) linked to the sushi domain of the IL-15Rα chain (IL15Rαsushi +IL15) fused in frame to the C'-terminus of a dimeric murine TGF-β-receptor (type II, TβRII) ectodomain-based ligand trap, termed FIST-15 (Fusion of Interleukin 15 with Sushi to TGF-β receptor). The rationale for the design of this protein is to prevent tumor-derived TGF-β from suppressing the immune response via the TGF-β ligand trap moiety, while simultaneously providing a potent stimulus for the activation of anti-tumor responses by an IL-15R agonist (IL-15Rαsushi +IL15).FIST-15 can neutralize TGF-β induced Smad signaling, and induce STAT3 and STAT5 phosphorylation by immunoblot and intracellular flow cytometric analysis of lymphocytes, suggesting that both protein domains are biochemically active. Functionally, FIST-15 is able to induce CD8+ T-cell proliferation at rates greater than IL-15 alone (CD8+ T-cell replicative index or fold-expansion of responding cells: 40, FIST-15, vs. 10, IL-15; p-value of unpaired T-test <0.05). The mitogenic effects of IL-15 are abrogated in CD8+ T-cells and NK cells in the presence of TGF-β. However, FIST-15 can overcome TGF-β mediated inhibition in both these cellular subsets (CD8+ T-cell replicative index: 20, FIST-15, vs. 5, IL-15, and NK cell replicative index: 40, FIST-15 vs. 5, IL-15; p-value <0.05).Rapid proliferation of the CD8+ central memory phenotype (CD62L+, CD44+) T-cells are seen with FIST-15 treatment. Compared to IL-15 expanded CD8+ T-cells, FIST-15 treatment also produced more IFN-γ, TNF-α, and IL-2 secreting CD8+ T-cells upon PMA/ionomycin stimulation. In addition to cytokines, production of anti-tumor effector molecules such as granzyme B is known to be inhibited by TGF-β. FIST-15 treated NK cells were superior to IL-15 treated NK cells in granzyme B production, even in the presence of TGF-β, as assayed by flow cytometric analysis (86.8% vs. 30.7% granzyme B expressing cells). Functionally, FIST-15 treated NK cells were also significantly more cytolytic against TGF-β secreting B16 murine melanoma cells in vitro compared to IL-15 treated NK cells (83.5% killing, FIST-15, vs. 24.4% killing, IL-15).C57Bl/6 mice with pre-established, syngeneic B16 melanoma tumors were treated with FIST-15 to assay the anti-tumor effects of the fusion protein in vivo. Mice receiving FIST-15 showed a significant delay in tumor growth (mean tumor volume: 345mm3) compared to control mice receiving conditioned media (mean tumor volume: 814.12mm3; p-value of paired T-test = 0.02) by day 21 post-tumor implantation. Furthermore, FIST-15 treated mice showed a significant survival advantage compared to control treated mice (80% vs 0%; p-value of log rank test = 0.0019) by day 27 post-tumor implantation. Mice immunized with B16 tumors transduced to express FIST-15 were also protected against subsequent wildtype B16 tumor challenge, suggesting that FIST-15 can trigger an adaptive immune response against tumor.Ongoing work utilizing FIST-15 in murine models of hematological malignancies, such as EL-4 lymphoma and C1498 AML, is currently underway. These models were selected due to their known overexpression of TGF-β isoforms that systemically inhibit endogenous anti-tumor responses, as well as the efficacy of immunotherapeutic agents. Indeed, many hematological malignancies acquire mutations that render them insensitive to the growth-inhibitory effects of TGF-β, where it may then be overexpressed as an oncogene to promote further tumor growth by inhibiting the immune system's anti-tumor capabilities (Dong et al Blood 2006). FIST-15 may present a viable immunotherapeutic strategy for hematological malignancies by combining the immune activating effects of IL-15 with the neutralization of immunosuppressive TGF-β. DisclosuresNg:Emory University: Patents & Royalties. Galipeau:Emory University: Patents & Royalties.

Size-dependent lymphatic uptake of nanoscale-tailored particles as tumor mass increases.

Aim:To investigate the size-dependent lymphatic uptake of nanoparticles in mice with rapidly growing syngeneic tumors.Materials & methods:Mice were inoculated subcutaneously with EL4 lymphoma cells and on day 5 or day 6 of tumor growth, injected peritumorally with either 29 nm or 58 nm of ultra-small superparamagnetic iron oxide nanoparticles. Twenty-four hours later the animals were imaged using MRI.Results & conclusion:The larger of the two particles can only be detected in the lymph node when injected in animals with 6-day-old tumors while the 29 nm ultra-small superparamagnetic iron oxide nanoparticle is observed on both time points. Tumor mass greatly impacts the size of particles that are transported to the lymph nodes.

Necrosis avid near infrared fluorescent cyanines for imaging cell death and their use to monitor therapeutic efficacy in mouse tumor models.

Quantification of tumor necrosis in cancer patients is of diagnostic value as the amount of necrosis is correlated with disease prognosis and it could also be used to predict early efficacy of anti-cancer treatments. In the present study, we identified two near infrared fluorescent (NIRF) carboxylated cyanines, HQ5 and IRDye 800CW (800CW), which possess strong necrosis avidity. In vitro studies showed that both dyes selectively bind to cytoplasmic proteins of dead cells that have lost membrane integrity. Affinity for cytoplasmic proteins was confirmed using quantitative structure activity relations modeling. In vivo results, using NIRF and optoacoustic imaging, confirmed the necrosis avid properties of HQ5 and 800CW in a mouse 4T1 breast cancer tumor model of spontaneous necrosis. Finally, in a mouse EL4 lymphoma tumor model, already 24 h post chemotherapy, a significant increase in 800CW fluorescence intensity was observed in treated compared to untreated tumors. In conclusion, we show, for the first time, that the NIRF carboxylated cyanines HQ5 and 800CW possess strong necrosis avid properties in vitro and in vivo. When translated to the clinic, these dyes may be used for diagnostic or prognostic purposes and for monitoring in vivo tumor response early after the start of treatment.

Tax peptide vaccine suppressed the leukemia in humanized mouse model of ATL

Cytotoxic T-lymphocyte (CTL) against HTLV-1 Tax has been demonstrated to play a vital role in controlling HTLV-1 infected cells in the infected carrier people. It was also shown that DNA immunization of mice with Tax expression vector suppressed the tumor growth of EL-4 lymphoma cells expressing Tax (EL4/Gax) through the activation of anti-Tax CTL [1]. Recently we established an HTLV-1-infected humanized mouse model (hu-NOG) to recapitulate ATL development and HTLV-1-specific immunity [2]. Here the efficacy of Tax-peptide vaccination was examined in the hu-NOG mouse model. Mixture of twelve overlapping peptides of 40-42 amino acids long encompassing whole Tax protein was inoculated subcutaneously three times weekly to hu-NOG mouse and then -irradiated HTLV-1 producing Jurkat cells were intraperitoneally injected to infect HTLV-1. While the control mice without immunization died of leukemia within two months of infection, Tax-immunization retarded the out growth of human lymphocytes after HTLV-1 infection and two out offive mice survived with alimited number of infected T-cells. IL-12 induction and the enhanced expression of Tax-specific CD8 T-cell were observed in the peripheral blood of immunized mice before infection. Furthermore, it was shown that the intranasal inoculation of Tax peptides has comparable effect on the onset of leukemia in the hu-NOG systems. These results suggest that the Tax peptide vaccination can elicit protective immunity against HTLV-1 infection and/or ATL developments.

Abstract 4201: Development of a syngeneic metastatic mouse model of malignant lymphoma

Abstract We have developed a mouse model of highly malignant lymphoma. The EL4 cell line was established from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. Red fluorescent protein (RFP)-EL4 lymphoma cells were established. EL4-RFP cancer cells were injected in the tail vein of C57/BL6 GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the superior mediastinum and in the liver 28 days later. The EL4-RFP tumors were visualized in the liver with the Olympus SZX7 microscope. Large EL4-RFP liver metastases in C57/BL6 GFP mice had GFP stromal cells derived from the host animal around the edge of EL4-RFP tumors. In addition, EL4-RFP lymphoma cells metastasis was formed in perigastric lymph node. EL4-RFP lymphoma cells circulated in the peripheral blood. Furthermore, EL4-RFP lymphoma cells were observed in bone marrow of C57/BL6 GFP transgenic mice. In summary, lymph node metastasis, liver metastasis and bone marrow metastasis were found approximately 4 weeks after transplantation. This malignant lymphoma model can be used to study early tumor development, metastasis, the role of stroma and discovery and evaluation of novel therapeuticus for this treatment-resistant disease. Citation Format: Takuro Matsumoto, Atsushi Suetsugu, Yuhei Shibata, Nobuhiko Nakamura, Hitomi Aoki, Takahiro Kunisada, Masahito Shimizu, Hisashi Tsurumi, Robert M. Hoffman. Development of a syngeneic metastatic mouse model of malignant lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2015-4201

Lymphatic dysfunction attenuates tumor immunity through impaired antigen presentation.

Tumor growth and metastasis of cancer involve autonomous tumor cell growth and host-tumor interactions. While tumor-specific immunity has been intensively studied in vitro, dynamic roles of lymphatic transport on tumor immunity in vivo have not been fully elucidated. In this study, we examined tumor growth and anti-tumor immune responses using kCYC mice, which demonstrate severe lymphatic dysfunction. Primary tumor growth was augmented in kCYC mice (compared to wild-type mice) when B16 melanoma or EL-4 lymphoma cells were subcutaneously injected. Expression of inflammatory cytokines such as IFN-γ, TNF-α, and IL-2 as well as IL-10 expression in draining lymph nodes (LNs) was significantly reduced in kCYC mice after tumor inoculation. Moreover, decreased levels of tumor-associated antigens were detected in draining LNs in kCYC mice, together with impaired antigen presentation. CD8+ T cells in draining LNs derived from kCYC mice bearing B16 melanoma also showed significantly decreased cytotoxic activity in vitro. Finally, tumor suppression activity of CD8+ T cells derived from kCYC mice bearing B16 melanoma was reduced when adoptively transferred to naive wild-type mice. In summary, these findings suggest that lymphatic transport is essential in generating optimal tumor-specific immune responses mediated by CD8+ T cells.

Histamine Promotes the Development of Monocyte-Derived Dendritic Cells and Reduces Tumor Growth by Targeting the Myeloid NADPH Oxidase

The efficiency of immune-mediated clearance of cancer cells is hampered by immunosuppressive mediators in the malignant microenvironment, including NADPH oxidase-derived reactive oxygen species. We aimed at defining the effects of histamine, an inhibitor of the myeloid NADPH oxidase/NOX2, on the development of Ag-presenting dendritic cells (DCs) from myeloid precursors and the impact of these mechanisms for tumor growth. Histamine was found to promote the maturation of human DCs from monocytes by increasing the expression of HLA-DR and costimulatory molecules, which resulted in improved induction of Th cells with Th0 polarity. Experiments using wild-type and NOX2-deficient myelomonoblastic cells showed that histamine facilitated myeloid cell maturation only in cells capable of generating reactive oxygen species. Treatment of mice with histamine reduced the growth of murine EL-4 lymphomas in parallel with an increment of tumor-infiltrating DCs in NOX2-sufficient mice but not in NOX2-deficient (gp91(phox) (-/-)) mice. We propose that strategies to target the myeloid NADPH oxidase may facilitate the development of endogenous DCs in cancer.

650. Development of Combination Therapy of Bifidobacterium-Based Oral Vaccine Displaying HCV-NS3 with Interferon-α

INTRODUCTION: Hepatitis C virus (HCV) is a major cause of chronic liver diseases, such as liver cirrhosis and hepatocellular carcinoma. The efficacy of the standard-of-care including interferon-α and ribavirin for chronic HCV infection is limited, especially in HCV genotype 1, the most prevalent genotype in Japan. It is considered that chronic HCV infection correlates strongly with failure of host immune response against HCV. Although HCV specific immune responses play an especially significant role in clearing virus, no effective vaccine against HCV was approved at present. We had successfully developed a recombinant Bifidobacterium based novel oral vaccine against HCV infection, displaying a HCV-nonstructural protein 3 (NS3) fusion peptide on the bacterial cell surface. We had found that this oral vaccine could induce HCV-NS3 specific CTL in mice. Then, in this study, to determine the therapeutic efficacy of this oral vaccine, the functional HCV specific cellular immune response was investigated by using HCV-NS3/4A antigen expressing tumor mouse model. Furthermore, to enhance the therapeutic efficacy, we combined interferon-α therapy with this oral vaccine in the mouse model. EXPERIMENTAL SUMMARY: Bifidobacterium longum (B. longum 2165) expressing NS3 fusion peptide derived HCV-genotype 1b was prepared and then heat inactivated for 5 min at 65 ° C for oral vaccination. For the tumor challenge, C57BL/6N mice were subcutaneously injected EL-4 lymphoma cells stably expressing HCV-NS3/4A, and then orally administered 2.4×108 CFU of inactivated B. longum 2165, active B. longum 2165, active B. longum transfected control vector (B. longum 2012), or PBS every other day for 4 weeks. In addition, for the combination therapy with interferon-α, inactivated B. longum 2165 and PBS groups were combined with 20,000 units of interferon-α subcutaneous injection every 4 days during vaccination. In the results, B. longum 2165 with interferon-α combination therapy significantly delayed tumor growth compared with the other treatments. For in vitro cytotoxicity assay, splenocytes were collected from immunized mice and restimulated with EL4-NS3/4A and IL-2. In the B. longum 2165 with interferon-α treated group, the cytotoxicity of splenocytes against EL4-NS3/4A was significantly higher than that of the other groups. CONCLUSION: This study demonstrated multiple oral vaccinations of B. longum 2165 could induce functional NS3 specific cellular immune responses in mice. Moreover, interferon-α combination therapy with the vaccine could strongly enhance the therapeutic efficacy of this vaccine. These findings support the feasibility of this therapeutic oral vaccine as additional agent of interferon based standard therapy against chronic HCV infection.

Denileukin diftitox reduces EL4 lymphoma growth by depleting regulatory T cells, which can be improved by αICOS (VAC12P.1116)

Abstract Immunotherapy shows promise in carcinoma treatments. As less is known about effects on lymphomas, we tested EL4 T cell lymphoma. We found that EL4 cells were CD25-CTLA4-B7H1loPD1hi by flow cytometry. Immune checkpoint blockade with αCTLA4, αB7H1, or αPD1 antibodies had no effect on tumor growth in EL4-challenged mice. αCD25 worked as prophylaxis (before EL4 challenge) but not therapy, despite depleting regulatory T cells (Tregs). In Foxp3DTR mice, specific Treg depletion reduced EL4 tumor growth, suggesting Treg depletion as a good treatment strategy. Denileukin diftitox (DD) is a relatively-specific Treg depletion drug with clinical benefits in mouse and human cancer. DD (5 μg/4 days starting 4 days after EL4 challenge) significantly delayed EL4 tumor growth and increased survival (P=.002) in WT mice. DD protection was lost in βδ TCR KO mice lacking all T cells, suggesting a T cell dependent treatment mechanism. In EL4-challenged WT, DD depleted Tregs &amp;gt; αCD25 but significantly increased ICOS by 2-fold on remaining Tregs &amp;gt; than on CD4+ or CD8+ T cells in tumor draining lymph nodes. Nearly all Tregs within tumors were ICOS+. ICOS+ Tregs were mostly CD44+ with hi Ki-67, suggesting activation that reduced DD treatment effects. In support, αICOS (100 μg/4 days starting 1 day after DD) enhanced DD-mediated EL4 tumor growth inhibition, but did not improve overall survival. DD-mediated Treg depletion plus αICOS and/or other adjuncts could lead to effective lymphoma immunotherapy.

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

The retinoic acid receptor-related orphan receptors α and γ (RORα and RORγ), are key regulators of helper T (Th)17 cell differentiation, which is involved in the innate immune system and autoimmune disorders. In this study, we investigated the effects of isoflavones on RORα/γ activity and the gene expression of interleukin (IL)-17, which mediates the function of Th17 cells. In doxycycline-inducible CHO stable cell lines, we found that four isoflavones, biochanin A (BA), genistein, formononetin, and daidzein, enhanced RORα- or RORγ-mediated transcriptional activity in a dose-dependent manner. In an activation assay of the Il17a promoter using Jurkat cells, these compounds enhanced the RORα- or RORγ-mediated activation of the Il17a promoter at concentrations of 1 × 10(-6)M to 1 × 10(-5)M. In mammalian two-hybrid assays, the four isoflavones enhanced the interaction between the RORα- or RORγ-ligand binding domain and the co-activator LXXLL peptide in a dose-dependent manner. In addition, these isoflavones potently enhanced Il17a mRNA expression in mouse T lymphoma EL4 cells treated with phorbol myristate acetate and ionomycin, but showed slight enhancement of Il17a gene expression in RORα/γ-knockdown EL4 cells. Immunoprecipitation and immunoblotting assays also revealed that BA enhanced the interaction between RORγt and SRC-1, which is a co-activator for nuclear receptors. Taken together, these results suggest that the isoflavones have the ability to enhance IL-17 gene expression by stabilizing the interactions between RORα/γ and co-activators. This also provides the first evidence that dietary chemicals can enhance IL-17 gene expression in immune cells.

Clever-1/stabilin-1 controls cancer growth and metastasis.

Immunosuppressive leukocytes and vasculature are important host cell components regulating tumor progression. Clever-1/Stabilin-1, a multifunctional scavenger and adhesion receptor, is constitutively present on a subset of type II macrophages and lymphatic endothelium, but its functional role in cancer is unknown. Here, we generated full Clever-1 knockout mice and cell-specific ones lacking Clever-1 either on macrophages or endothelium. We also used anti-Clever-1 antibody therapy to treat B16 melanoma and EL-4 lymphoma. Clever-1-deficient mice had smaller primary and metastatic tumors than wild-type (WT) controls. Growth of primary tumors, but not of metastases, was attenuated also in mice lacking Clever-1 selectively in macrophages or in vascular endothelium. Anti-Clever-1 antibody treatment inhibited tumor progression in WT mice. Both genetically and therapeutically induced absence of functional Clever-1 led to diminished numbers of immunosuppressive leukocyte types in tumors. Functionally Clever-1 mediated binding of immunosuppressive leukocytes to the intratumoral blood vessels aberrantly expressing Clever-1, and tumor cell traffic via the lymphatics. The antibody therapy did not aggravate autoimmunity. This work identifies Clever-1 in type II macrophages and in tumor vasculature as a new immunosuppressive molecule in cancer. Our finding that Clever-1 supports binding of tumor-infiltrating lymphocytes to tumor vasculature increases our understanding of leukocyte immigration to tumors. The ability of anti-Clever-1 antibody treatment to attenuate tumor progression in WT mice in vivo is therapeutically relevant. Thus, Clever-1 may be an emerging new target for modulating immune evasion and lymphatic spread in cancer.

Adiposity As a Principal Component of Lethal Cytokine Storm Following Cancer Immunotherapy in Aged Mice

The use of immunotherapy (IT) in cancer has recently resulted in impressive responses. Yet, their usages, especially those involving cytokine therapies, have also resulted in the induction of severe systemic toxicities often not previously characterized in their preclinical animal studies. Our laboratory has demonstrated that treatment of EL4 lymphoma with a combination immunotherapy consisting of monoclonal agonistic antibody CD40 and IL-2 resulted in significant anti-tumor responses leading to overall increases in survival using young inbred mice (2-6 months of age). Yet, the majority of hematological cancers occur in aged individuals, with most diagnoses occurring past the age of 60. We now demonstrate that systemic immunotherapy administration in aged (>16 months of age), but not young, mice resulted in the induction of a rapid cytokine release syndrome, also known as “cytokine storm”, culminating in multi-organ damage (liver, lung, and gut) leading to rapid lethality by day 2 of treatment. Aging is accompanied by an overall redistribution of body mass, with a decrease in lean muscle mass and increase in adiposity. Similarly, we found that normal aging in inbred mice housed under specific pathogen free conditions was accompanied by increases in visceral fat that was similar to young obese (ob/ob or diet-induced obese [DIO]) mice. We therefore assessed the impact of aging and obesity on inflammatory responses to cancer immunotherapeutics. We determined the effects of increased body fat on systemic immunotherapy tolerance in aged mice compared to young obese mice. Both young ob/ob and DIO generated pro-inflammatory cytokine (ie TNFa and IL6) levels and organ pathologies comparable to aged WT mice following immunotherapy, culminating in rapid lethality after several days of therapy. We observed that young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNFa+ macrophages in comparison to young lean mice with immunotherapy. Administration of aCD40/IL-2 with macrophage depletion or TNF-blockade prevented the development of cytokine storms within young obese mice, providing protection from lethality, suggesting that the toxicity was macrophage mediated through increases in TNFa. Calorie-restricted aged mice contain less visceral fat and upon aCD40/IL-2 administration displayed reduced cytokine levels, protection from organ pathology, and protection from lethality. Our data demonstrates adiposity as a critical factor in the age-associated inflammatory pathologic responses to systemic anti-cancer immunotherapy and may have a significant impact when immunotherapy is used clinically within cancer therapy regimens. These data also underscore the critical importance of taking aging and body fat into consideration with preclinical assessments. DisclosuresNo relevant conflicts of interest to declare.

Functional consequences of TCR transgenic β-chain expression (TUM2P.908)

Abstract We have generated mouse strain expressing transgenic β-chain of TCR 1D1 (belonging to the Vβ6 family) on the genetic background B10.D2(R101) (KdIdDb). The repertoire of peripheral T lymphocytes in these mice contains 70-80% of T cells expressing transgenic β-chain and 20-30% of T cells expressing endogenous β-chains. Expression of transgenic β-chain resulted in T cells with phenotype CD44-CD62L+ characteristic of “naïve” T lymphocytes. On the contrary, CD8+ T lymphocytes expressing endogenous β-chains had surface phenotype of activated effectors CD44+CD62L-. In wild-type B10.D2(R101) mice, immunization with lymphoma EL4 (H-2b) cells induced a vigorous CTL response targeted to the H-2Kb molecule and resulted in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in MLR and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, mostly CD8+ T-lymphocytes with endogenous β-chains became CD44+CD62L- effectors in transgenic mice, whereas only a small part of the T-lymphocytes expressing transgenic β-chains became effectors. Effectors CD8+CD44+CD62L- expressing endogenous β-chains showed enhanced expression of PD-1 and contained increased percent of KLRG1-positive cells. The acquisition of these characteristic features was associated with immune selection of H-2Kb-negative EL4 cells and subsequent tumor escape.

Radiotherapy-Induced Anti-Tumor Immunity Contributes to the Therapeutic Efficacy of Irradiation and Can Be Augmented by CTLA-4 Blockade in a Mouse Model

PurposeThere is growing evidence that tumor-specific immune responses play an important role in anti-cancer therapy, including radiotherapy. Using mouse tumor models we demonstrate that irradiation-induced anti-tumor immunity is essential for the therapeutic efficacy of irradiation and can be augmented by modulation of cytotoxic T lymphocyte (CTL) activity.Methods and MaterialsC57BL/6 mice, syngeneic EL4 lymphoma cells, and Lewis lung carcinoma (LL/C) cells were used. Cells were injected into the right femurs of mice. Ten days after inoculation, tumors were treated with 30 Gy of local X-ray irradiation and their growth was subsequently measured. The effect of irradiation on tumor growth delay (TGD) was defined as the time (in days) for tumors to grow to 500 mm3 in the treated group minus that of the untreated group. Cytokine production and serum antibodies were measured by ELISA and flow cytometry.ResultsIn the EL4 tumor model, tumors were locally controlled by X-ray irradiation and re-introduced EL4 cells were completely rejected. Mouse EL4-specific systemic immunity was confirmed by splenocyte cytokine production and detection of tumor-specific IgG1 antibodies. In the LL/C tumor model, X-ray irradiation also significantly delayed tumor growth (TGD: 15.4 days) and prolonged median survival time (MST) to 59 days (versus 28 days in the non-irradiated group). CD8(+) cell depletion using an anti-CD8 antibody significantly decreased the therapeutic efficacy of irradiation (TGD, 8.7 days; MST, 49 days). Next, we examined whether T cell modulation affected the efficacy of radiotherapy. An anti-CTLA-4 antibody significantly increased the anti-tumor activity of radiotherapy (TGD was prolonged from 13.1 to 19.5 days), while anti-FR4 and anti-GITR antibodies did not affect efficacy.ConclusionsOur results indicate that tumor-specific immune responses play an important role in the therapeutic efficacy of irradiation. Immunomodulation, including CTLA-4 blockade, may be a promising treatment in combination with radiotherapy.