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Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers and is associated with a worse clinical outcome. This poor clinical outcome is attributed to a lack of defined targets and patient-to-patient heterogeneity in target antigens and immune responses. To address such heterogeneity, we tested the efficacy of a personalized vaccination approach for the treatment of TNBC using the 4T1 murine TNBC model. We isolated tumor membrane vesicles (TMVs) from homogenized 4T1 tumor tissue and incorporated glycosyl phosphatidylinositol (GPI)-anchored forms of the immunostimulatory B7-1 (CD80) and IL-12 molecules onto these TMVs to make a TMV vaccine. Tumor-bearing mice were then administered with the TMV vaccine either alone or in combination with immune checkpoint inhibitors. We show that TMV-based vaccine immunotherapy in combination with anti-CTLA-4 mAb treatment upregulated immunomodulatory cytokines in the plasma, significantly improved survival, and reduced pulmonary metastasis in mice compared to either therapy alone. The depletion of CD8(+) T cells, but not CD4(+) T cells, resulted in the loss of efficacy. This suggests that the vaccine acts via tumor-specific CD8(+) T cell immunity. These results suggest TMV vaccine immunotherapy as a potential enhancer of immune checkpoint inhibitor therapies for metastatic triple-negative breast cancer.

Imaging techniques based on fluorescence and bioluminescence have been important tools in visualizing tumor progression and studying the effect of drugs and immunotherapies on tumor immune microenvironment in animal models of cancer. However, transgenic expression of foreign proteins may induce immune responses in immunocompetent syngeneic tumor transplant models and augment the efficacy of experimental drugs. In this study, we show that the growth rate of Lewis lung carcinoma (LL/2) tumors was reduced after transduction of tdTomato and luciferase (tdTomato/Luc) compared to the parental cell line. tdTomato/Luc expression by LL/2 cells altered the tumor microenvironment by increasing tumor-infiltrating lymphocytes (TILs) while inhibiting tumor-induced myeloid-derived suppressor cells (MDSCs). Interestingly, tdTomato/Luc expression did not alter the response of LL/2 tumors to anti-PD-1 and anti-CTLA-4 antibodies. These results suggest that the use of tdTomato/Luc-transduced cancer cells to conduct studies in immune competent mice may lead to cell-extrinsic tdTomato/Luc-induced alterations in tumor growth and tumor immune microenvironment that need to be taken into consideration when evaluating the efficacy of anti-cancer drugs and vaccines in immunocompetent animal models.

Metastatic triple-negative breast cancer (TNBC) has a poor prognosis primarily due to metastatic potential and resistance to currently available therapies. Recent studies suggest that TNBC may be a viable target for immunotherapy due to the presence of high levels of infiltrating lymphocytes. However, many TNBC patients do not respond due to high intra-tumoral and inter-patient heterogeneity. Utilizing a novel protein transfer method, we generate a personalized vaccine based on tumor membrane vesicles (TMVs) derived from whole tumor tissues modified with glycolipid-anchored forms of the immunostimulatory molecules (GPI-ISMs) B7-1 and IL-12. In prior studies we observed that simultaneous delivery of TMVs along with biological adjuvants enhanced CD8 T cell immunity. Herein, we investigated the impact of standard-of-care (SOC) chemotherapy on the resulting quality of the TMV vaccine. Mice were injected with 4T1 TNBC cells subcutaneously and treated with three rounds of SOC drugs starting at day 15. Chemotherapies affected tumor growth to varying degrees. Mice were euthanized and tumors were harvested for TMV preparation. Results show that depending on the treatment, the yield and particle size of TMVs was impacted. While SOC drugs affected some surface markers on TMVs, expression of CD24 and CD11b was unchanged. Further, there was reduced uptake of TMVs by dendritic cells prepared from cisplatin and 5-fluorouracil treated tumors. GPI-ISMs were equally incorporated by protein transfer onto TMVs despite SOC treatments. In summary, our studies in the 4T1 model show that a TMV vaccine can be successfully prepared after chemotherapy, but the impact on TMV yield and size justifies further investigation. Supported by R01 CA202763

Immune checkpoint inhibitor (ICI) immunotherapy improved the survival of head and neck squamous cell carcinoma (HNSCC) patients. However, more than 80% of the patients are still resistant to this therapy. To test whether the efficacy of ICI therapy can be improved by vaccine-induced immunity, we investigated the efficacy of a tumor membrane-based vaccine immunotherapy in murine models of HNSCC. The tumors, grown subcutaneously, are used to prepare tumor membrane vesicles (TMVs). TMVs are then incorporated with glycolipid-anchored immunostimulatory molecules GPI-B7-1 and GPI-IL-12 by protein transfer to generate the TMV vaccine. This TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. The tumor-free mice survived for several months, remained tumor-free, and were protected following a secondary tumor cell challenge, suggesting that the TMV vaccine induced an anti-tumor immune memory response. However, no synergy with anti-PD1 mAb was observed in this model. In contrast, the TMV vaccine was effective in inhibiting MOC1 and MOC2 murine oral cancer models and synergized with anti-PD1 mAb in extending the survival of tumor-bearing mice. These observations suggest that tumor tissue based TMV vaccines can be harnessed to develop an effective personalized immunotherapy for HNSCC that can enhance the efficacy of immune checkpoint inhibitors.

BackgroundPD-L1 is one of the major immune checkpoints which limits the effectiveness of antitumor immunity. Blockade of PD-L1/PD-1 has been a major improvement in the treatment of certain cancers, however, the response rate to checkpoint blockade remains low suggesting a need for new therapies. Metformin has emerged as a potential new drug for the treatment of cancer due to its effects on PD-L1 expression, T cell responses, and the immunosuppressive environment within tumors. While the benefits of metformin in combination with checkpoint blockade have been reported in animal models, little remains known about its effect on other types of immunotherapy.MethodsVaccine immunotherapy and metformin were administered to mice inoculated with tumors to investigate the effect of metformin and TMV vaccine on tumor growth, metastasis, PD-L1 expression, immune cell infiltration, and CD8 T cell phenotype. The effect of metformin on IFN-γ induced PD-L1 expression in tumor cells was assessed by flow cytometry, western blot, and RT-qPCR.ResultsWe observed that tumors that respond to metformin and vaccine immunotherapy combination show a reduction in surface PD-L1 expression compared with tumor models that do not respond to metformin. In vitro assays showed that the effect of metformin on tumor cell PD-L1 expression was mediated in part by AMP-activated protein kinase signaling. Vaccination results in increased T cell infiltration in all tumor models, and this was not further enhanced by metformin. However, we observed an increased number of CD8 T cells expressing PD-1, Ki-67, Tim-3, and CD62L as well as increased effector cytokine production after treatment with metformin and tumor membrane vesicle vaccine.ConclusionsOur data suggest that metformin can synergize with vaccine immunotherapy to augment the antitumor response through tumor-intrinsic mechanisms and also alter the phenotype and function of CD8 T cells within the tumor, which could provide insights for its use in the clinic.

Dendritic cells (DCs) are the most effective antigen presenting cells for the development of T cell responses. The only FDA approved DC-based immunotherapy to date is Sipuleucel-T, which utilizes a fusion protein to stimulate DCs ex vivo with GM-CSF and simultaneously deliver the antigen PAP for prostate cancer. This approach is restricted by the breadth of immunity elicited to a single antigen, and to cancers that have a defined tumor associated antigen. Other multi-antigen approaches have been restricted by poor efficacy of vaccine adjuvants. We have developed a vaccine platform that consists of autologous DCs pulsed with cytokine-adjuvanted tumor membrane vesicles (TMVs) made from tumor tissue, that encapsulate the antigenic landscape of individual tumors. Here we test the efficacy of DCs pulsed with TMVs incorporated with glycolipid-anchored immunostimulatory molecules (GPI-ISMs) in HER2-positive and triple negative breast cancer murine models. Pulsing of DCs with TMVs containing GPI-ISMs results in superior uptake of vesicles, DC activation and cytokine production. Adaptive transfer of TMV-pulsed DCs to tumor bearing mice results in the inhibition of tumor growth, reduction in lung metastasis, and an increase in immune cell infiltration into the tumors. These observations suggest that DCs pulsed with TMVs containing GPI-GM-CSF and GPI-IL-12 can be further developed to be used as a personalized immunotherapy platform for cancer treatment.

Breast cancer is the leading cause of death among female cancer patients. Our lab has developed a therapeutic cancer vaccine approach for triple negative breast cancer (TNBC). In this study, we investigated whether vaccine developed from a human TNBC cell line could effectively induce antitumor immunity, which control the growth of tumors in humanized mice implanted with the human TNBC cell line. Human CD34+ HSC implanted NSG-SGM3 from JAX labs and NOG-EXL mice from Taconic Biosciences used for the studies. Humanized NSG-SGM3 mice were vaccinated with 100 mg of the vaccine once every 2 weeks (3 doses). Control mice were injected with vehicle (PBS). One week after the last vaccination, mice were challenged with MDA-MB-231 TNBC cells subcutaneously in the hind flank. Body weight and tumor growth were monitored. All the mice in both groups have developed tumors, splenomegaly and metastatic lesions in the liver, lungs and spleen. However, all the mice also developed GvHD and need to be euthanized before completion of the study. We have also observed an allo-response (CD69+ T cells in control mice with MDA-MB-231 tumors) to the HLA-matched tumor cells but the tumors eventually developed into metastatic disease affecting multiple organs (liver, lungs and spleen) suggesting that the allo-response is not effective against the tumor. Since the tumor-bearing mice developed GvHD and becoming anemic even in a specific pathogen-free barrier facility, future studies will need to be tested in humanized mice which do not develop GvHD and live longer for carrying out vaccine immunotherapy studies. Funding: NIH/NCI R01 CA202763 (PS & CP).

Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine (TMV) immunotherapy in murine SCCHN models. The MOC1, MOC2 and SCC VII tumors grown subcutaneously in syngeneic mice were harvested to generate TMVs. TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules GPI-B7.1 and GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine after tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. The TMV vaccine inhibited tumor growth and improved the survival of mice challenged with MOC1 or SCC VII tumor cells. Interestingly, combination of anti-PD1 antibody with TMV vaccine decreased MOC1 tumor growth. However, MOC2 (poorly immunogenic tumor) did not respond to anti-PD1 or anti-CTLA-4 antibody therapy but significantly inhibited by TMV vaccine. MOC2 cells metastasize to the lungs in control mice but it is significantly inhibited in mice administered with TMV vaccine. TMV vaccine increased the infiltration of CD4+ and CD8+ T cells into the tumors suggesting that TMV vaccine converts cold tumors into hot tumors. These observations suggest that TMV vaccines can be harnessed to develop an effective immunotherapy for squamous cell carcinoma of the head and neck.

Immunotherapy has emerged as a promising treatment of breast cancer, however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by promoting CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T cell infiltration to the tumor, and combination with metformin caused a decrease in the accumulation of T cells, despite having better control of tumor growth. Further, we show that metformin can reduce PD-L1 expression within the tumor microenvironment and result in more IFN-γ producing CD8 T cells within the tumor, providing a mechanistic insight for the improvement of TMV-based vaccine immunotherapy.

Immunotherapy has emerged as a promising treatment of breast cancer; however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an antitumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert antitumor effects by promoting CD8 T-cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T-cell infiltration to the tumor, and combination with metformin caused a decrease in the accumulation of T cells, despite having better control of tumor growth. Further, we show that metformin can reduce PD-L1 expression within the tumor microenvironment and result in more IFN-γ-producing CD8 T cells within the tumor, providing a mechanistic insight for the improvement of TMV-based vaccine immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley L. Huang, Christopher D. Pack, Sampath Ramachandiran, Periasamy Selvaraj. Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine-generated CD8 T cells in a murine model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B32.

Lung cancer is the second most common cancer among men and women and the leading cause of cancer deaths in the world. Despite the advancements in diagnostic technologies and the development of novel treatments in the recent years such as immune checkpoint inhibitor (ICI) therapy, the prognosis of lung cancer patients is still poor, and the survival rate is low. The patient-to-patient heterogeneity is the major problem in treating lung cancer, which results in differential responses to ICI therapy. In this study, we investigated the efficacy of a vaccine immunotherapy prepared by modification of tumor membrane vesicles (TMVs) prepared from the tumors to express immunostimulatory molecules in combination with ICI therapy in a partially ICI resistant murine lung cancer model. We demonstrate that prophylactic vaccination of TMV adjuvanted with immunostimulatory molecules prevents the CMT-167 tumor growth in mice and helps the mice develop immunological memory to the tumor cell challenge. In a therapeutic setting, the TMV vaccine inhibited tumor growth and induced primary tumor regression in 40% of mice and significantly reduced the metastatic tumor burden. However, the combination of TMV vaccination with anti-PD-1 mAb doesn’t have a synergistic or additive effect on the primary or metastatic tumor growth. Overall, these studies provide insight into using the TMV vaccine immunotherapy for treating lung cancer. Funding: Supported by the Lung Cancer seed grant from the Winship Cancer Institute of Emory University Citation Format: Lei Huang, Dr. Selvaraj's lab. Immunotherapy with TMV vaccine inhibits metastatic tumor growth in an immune checkpoint resistant murine lung cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-202.

Breast cancer remains the most commonly diagnosed cancers among women, with over 250,000 new cases per year in the United States. Immunotherapy has emerged as a promising treatment of breast cancer, however response rates to therapy remain far from ideal. In this study, we investigated the combinatorial effect of tumor membrane vesicle (TMV)-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by maintaining CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while combination with metformin resulted in greater inhibition of primary growth. Importantly, TMV vaccination reduced the metastatic burden in the lungs while combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T cell infiltration to the tumor, while combination with metformin caused a decrease in the accumulation of T cells, despite having better outcomes. Further, we show that metformin is capable of reducing PD-L1 expression within the tumor microenvironment in both tumor cells as well as myeloid-derived suppressor cells, providing insight for the improvement of TMV-based immunotherapy. Citation Format: Luis E. Munoz, Ramireddy Bommireddy, Haley Huang, Janet Kim, Periasamy Selvaraj. Metformin enhances the efficacy of tumor membrane vesicle (TMV)-based vaccine immunotherapy in a murine model of breast [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-203.

Introduction: Head and neck cancer is a leading cause of cancer related deaths accounting for approximately 3% of all cancer related mortalities in the US. Currently, there is no cure for the advanced squamous cell carcinoma of the head and neck (SCCHN) thus development of efficacious therapies is urgently needed. To test whether vaccine-induced immunity inhibits tumor growth, we investigated efficacy of a tumor membrane-based vaccine immunotherapy in a murine SCCHN (SCC VII) model. Materials and Methods: The SCC VII tumors grown subcutaneously in C3H/HeJ mice were harvested to generate tumor membrane vesicles (TMVs). TMVs were then protein transferred with glycolipid-anchored immunostimulatory molecules mouse GPI-B7.1 and mouse GPI-IL-12 to generate the TMV vaccine. Mice were vaccinated with TMV vaccine either before (prophylactic) or after SCC VII tumor cell challenge (therapeutic) and tumor growth was monitored every 3 days. Survival was then assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Results: SCCVII cells express MHC I, CD44, CD47 and respond to IFN-γ in vitro. The therapeutic TMV vaccine inhibited tumor growth and improved the survival of mice challenged with SCCVII tumor cells. Tumor-free mice remained protective from rechallenge with SCCVII cells. Conclusions: These observations suggest that tumor tissue-based vaccines can be harnessed to develop an effective immunotherapy for SCCHN. Funding: Supported by Head and Neck SPORE pilot funding from Emory Winship Cancer Institute. Citation Format: Ramireddy Bommireddy, Luis Munoz, Chrstopher D. Pack, Sampath Ramachandiran, Shaker JC Reddy, Janet Kim, Georgia Chen, Nabil F. Saba, Dong M. Shin, Periasamy Selvaraj. TMV vaccine inhibits growth of squamous cell carcinoma of head and neck in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4097.

e15183 Background: Current immunotherapies fail to benefit patients with colon cancer, largely due to high molecular heterogeneity and low frequencies of tumor infiltrating lymphocytes. In this study, we are addressing this limitation via a novel technology to expand patient tumor specific T cells ex-vivo, using patient’s matched tumor and peripheral blood mononuclear cells (PBMCs). This technology utilizes tumor membrane vesicles (TMVs) made from excised tumor tissue, as a source of tumor associated antigens, and further modified to express IL-12 and B7-1 for ex-vivo expansion of tumor specific T cells. Additionally, the T cells are cultured with PI3K delta inhibitor (idelalisib) and vasoactive intestinal peptide antagonist (VIPhyb) to increase yield, polyfunctionality and in-vivo persistence. Methods: Matched tumor and blood were collected from consented colon cancer patients. TMVs were prepared from excised tumors by homogenizing and centrifuging the tumor over a sucrose gradient. TMVs were then made to express glycosyl phosphatidylinositol (GPI) linked IL-12 and B7-1 (decorated TMV) via a proprietary protein transfer technology. PBMCs isolated from matched blood were cultured in media with 10ug/ml decorated TMV with/without VIPhyb(3uM) and idelalisib (100nM) for 14 days. T cells were then phenotyped for percentage of IFN gamma secreting CD4+ and CD8+ T cells and injected into PDX mice bearing matched tumors (2 doses of 1 million and 4 million cells, 20 days apart). Results: 14 days after expansion, T cells expanded with decorated TMVs+VIPhyb+idelalisib (TMVs+drugs) resulted in significantly increased number of IFN gamma secreting CD8+ T cells (25% of T cells), when compared to T cells treated with decorated TMVs without VIPhyb and idelalisib (13% of T cells). Further, when injected to PDX mice bearing matched tumors, and followed for differences in tumor growth, we observed that on day 30 after T cell injection, while the tumor volumes increased 4.5-fold in the untreated group, the tumors in the TMVs+drugs group only increased 2.8-fold. Conclusions: In this study, we have thus developed a novel technology to expand patient tumor specific T cells ex-vivo, for adoptive T cell therapy against metastatic colon cancer.

Adoptive T cell therapy in solid tumors such as colorectal cancers are challenging due the low frequency of tumor infiltrating T cells and high molecular tumor heterogeneity. In this study, we are proposing to address this issue by developing a novel technology to expand patient tumor specific T cells ex-vivo, using patient’s matched tumor and PBMCs. Published data from our group shows T cells isolated from heavily treated lymphoma patients when treated with inhibitors of PI3K delta (idelalisib) and vasoactive intestinal peptide (VIPhyb) signaling results in decreased levels of senescent T cells and increased persistence in-vivo in NSG mice. Additionally, we have demonstrated that tumor membrane vesicles (TMVs) prepared from human tumor cells decorated with IL-12 and B7-1 (decorated TMVs), stimulate expansion of tumor antigen specific T cells with increased IFN gamma release. This study thus combines both strategies to obtain tumor antigen specific T cells with enhanced anti-tumor activity and in-vivo persistence. Preliminary data was generated with PBMCs obtained from consented colon cancer patients expanded with decorated TMVs with/without VIPhyb and idelalisib. Results showed that on day 14 of T cell expansion, T cells expanded with decorated TMVs + VIPhyb + idelalisib resulted in significantly increased number of IFN gamma secreting CD8+ T cells (25% of T cells), when compared to T cells treated with decorated TMVs without VIPhyb and idelalisib (13% of T cells). On the other hand, in the absence of decorated TMVs, there were less IFN gamma secreting T cells, both in the CD4 and CD8 compartment. Further experiments are underway to test the persistence and efficacy of the expanded T cells in PDX mice with matched human colon cancer.

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies lung cancer models LL/2 and CMT-167 models. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then modified with immunostimulatory molecules and used for immunization. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of CMT-167 was assessed by clonogenic assay. TMV vaccine alone, or in combination, anti-PD-1 or anti-CTLA-4 antibodies neither decreased the tumor growth nor improved the survival of mice challenged with LL/2 tumor cells. The prophylactic vaccination of TMV vaccine prevents the CMT-167 tumor growth in mice and helps the mice develop immunological memory to the tumor cell challenge. In a therapeutic setting, the TMV vaccine inhibited tumor growth and induced primary tumor regression in 40% of mice and significantly reduced the metastatic tumor burden. Anti-tumor immunity induced by TMV vaccine requires CD4+ and NK cells albeit to a lesser extent but not CD8+ T cells for controlling the tumor growth. These models provide a valuable tool in analyzing the mechanism of interaction of immune cells with tumors that lead to either tumor regression or resistance to immunotherapies.

The development of colorectal cancer in the azoxymethane-induced mouse model can be observed by using a miniaturized optical coherence tomography (OCT) imaging system. This system is uniquely capable of tracking disease development over time, allowing for the monitoring of morphological changes in the distal colon due to tumor development and the presence of lymphoid aggregates. By using genetically engineered mouse models deficient in Interleukin 6 (IL-6) and Smad family member 3 (Smad3), the role of inflammation on tumor development and the immune system can be elucidated. Smad3 knockout mice develop inflammatory response, wasting, and colitis associated cancer while deficiency of proinflammatory cytokine IL-6 confers resistance to tumorigenesis. We present pilot data showing that the Smad3 knockout group had the highest tumor burden, highest spleen weight, and lowest thymus weight. The IL-6 deficiency in Smad3 knockout mice prevented tumor development, splenomegaly, and thymic atrophy. This finding suggests that agents that inhibit IL-6 (e.g. anti-IL-6 antibody, non-steroidal anti-inflammatory drugs [NSAIDs], etc.) could be used as novel therapeutic agents to prevent disease progression and increase the efficacy of anti-cancer agents. OCT can also be useful for initiating early therapy and assessing the benefit of combination therapy targeting inflammation.

Calcineurin (CN) is a phosphatase that activates nuclear factor of activated T cells (NFAT). While the CN inhibitors cyclosporine A (CsA) and tacrolimus (FK506) can prevent graft rejection, they also cause inflammatory diseases. We investigated the role of calcineurin using mice deficient in the CN catalytic subunit Aβ (CNAβ). Cnab(-/-) mice exhibit defective thymocyte maturation, splenomegaly and hepatomegaly. Further, as Cnab(-/-) mice age, they exhibit spontaneous T-cell activation and enhanced production of proinflammatory cytokines (IL-4, IL-6, and IFNγ). FOXP3(+) T(reg) cells were significantly decreased in Cnab(-/-) mice likely contributing to increased T-cell activation. Interestingly, we found that CNAβ is critical for promotion of BCL-2 expression in FOXP3(+) T(reg) and for permitting TGFβ signaling, as TGFβ induces FOXP3 in control but not in Cnab(-/-) T-cells. Together, these data suggest that CNAβ is important for the production and maintenance of T(reg) cells and to ensure mature T-cell quiescence.

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies in an aggressive, non-metastatic, immune checkpoint inhibitor resistant lung cancer model (LL/2) and a highly metastatic lung cancer (CMT-167) model. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then mixed with adjuvants and used for immunization in conjunction with immune checkpoint inhibitors. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of CMT-167 was assessed by clonogenic assay. TMV alone, or in combination with adjuvants, anti-PD-1 or anti-CTLA-4 antibodies neither decreased the tumor growth nor improved the survival of mice challenged with LL/2 tumor cells. However, anti-PD-1 antibody delayed the tumor growth, improved the survival and reduced pulmonary metastasis in mice challenged with CMT-167 tumor cells. These models provide a valuable tool in analyzing the mechanism of interaction of immune cells with tumors that lead to either tumor regression or resistance to immunotherapies.

Breast cancer remains the most commonly diagnosed cancers among American women, with over 250,000 new cases expected per year. Breast cancer can be subdivided into different categories depending on the expression of receptors, most commonly into ER+, PR+, HER2+, and TNBC. Clinically, small molecule inhibitors of the hormone receptors and antibodies for HER2 have been developed to target these receptors and inhibit tumor signaling or eliminate these cells via phagocytosis and ADCC. Despite this, tumor cells that develop resistance to therapy remain a significant unmet need as they tend to be more aggressive and metastasize. We developed a novel immunotherapy approach that addresses the high degree of inter-patient and intra-tumor heterogeneity by making tumor membrane vesicle (TMV) vaccines out of individual tumor samples and modifying them with GPI-linked molecules like IL-12 and B7-1 to induce an antitumor immune response. In the present study, we investigated whether GPI-GM-CSF and GPI-IL-12 on TMVs from HER2+ murine breast cancer can target and activate antigen presenting cells. Here, we show that TMVs containing with GPI-GM-CSF and GPI-IL-12 can be phagocytosed by both macrophages and dendritic cells, induce upregulation of MHC-I, MHC-II and CD86, and inflammatory cytokine production in vitro. Further, when the DCs activated with our TMV vaccine were co-cultured with syngeneic T cells, they induced the upregulation of T cell activation markers. In summary, our results indicate that TMVs containing GPI-IL-12 and GPI-GM-CSF can enhance uptake and maturation of APCs and induce a strong T cell response downstream making it a potential therapeutic approach for HER2+ breast cancer.

Transforming growth factor (TGF) beta1) is an immunoregulatory cytokine involved in self-tolerance and lymphocyte homeostasis. Tgfb1 knock-out (KO) mice develop severe multi-focal autoimmune inflammatory lesions due to [Ca(2+)]i deregulation in T cells, and die within 3 weeks after birth. Because the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1(-/-) thymocytes, and because calcineurin Abeta (CNAbeta)-deficient mice do not reject allogenic tumours, we have generated Tgfb1(-/-) Cnab(-/-) mice to address whether CNAbeta deficiency prevents T cell activation and inflammation in Tgfb1(-/-) mice. Here we show that in Tgfb1(-/-) Cnab(-/-) mice inflammation is reduced significantly relative to that in Tgfb1(-/-) mice. However, both CD4(+) and CD8(+) T cells in double knock-out (DKO) mice are activated, as revealed by up-regulation of CD11a lymphocyte function-associated antigen-1 (LFA-1), CD44 and CD69 and down-regulation of CD62L. These data suggest that deficiency of CNAbeta decreases inflammatory lesions but does not prevent activation of autoreactive T cells. Also Tgfb1(-/-) T cells can undergo activation in the absence of CNAbeta, probably by using the other isoform of calcineurin (CNAalpha) in a compensatory manner. CNAbeta-deficient T cells undergo spontaneous activation in vivo and are activated upon anti-T cell receptor stimulation in vitro. Understanding the role of calcineurin in T cell regulation should open up new therapeutic opportunities for inflammation and cancer.

TGFbeta1 is considered to be required for peripheral maintenance of CD4(+)CD25(+)FOXP3(+) T(reg) cells. However, we demonstrate no reduction in the percentage of such T cells in the spleens and thymi of Tgfb1(-/-) mice. Although putative T(reg) cells, characterized as CD4(+)CD25(+)FOXP3(+)CD62L(+) T cells, are increased in Tgfb1(-/-) mice, they may be inadequate to control activated T cells since the ratio of activated T cells:putative T(reg) cells is several-fold higher in Tgfb1(-/-) mice than in control mice. We further show that whereas Tgfb1(-/-) mice that express a chicken OVA-specific TCR transgene (DO11.10) have an increase in putative T(reg) cells, there are no detectable CD4(+)CD25(+) T cells in the spleens of DO11.10 Rag1(-/-) mice suggesting that T(reg)-cell generation is self-antigen dependent regardless of whether they express Tgfb1. Finally, we demonstrate that Tgfb1(-/-) T cells remain responsive to the suppressive effect of TGFbeta1 in vitro. These data suggest that TGFbeta1 is required for the regulatory function of T(reg) cells to prevent activation of T cells and autoimmunity.

Ample evidence suggests a role of TGF-β in preventing autoimmunity. Multiorgan inflammatory disease, spontaneous activation of self-reactive T cells, and autoantibody production are hallmarks of autoimmune diseases, such as lupus. These features are reminiscent of the immunopathology manifest in TGF-β1-deficient mice. In this study, we show that lupus-prone (New Zealand Black and White)F1 mice have reduced expression of TGF-β1 in lymphoid tissues, and TGF-β1 or TGF-β1-producing T cells suppress autoantibody production. In contrast, the expression of TGF-β1 protein and mRNA and TGF-β signaling proteins (TGF-β receptor type II and phosphorylated SMAD3) increases in the target organs, i.e., kidneys, of these mice as they age and develop progressive organ damage. In fact, the levels of TGF-β1 in kidney tissue and urine correlate with the extent of chronic lesions that represent local tissue fibrosis. In vivo TGF-β blockade by treatment of these mice with an anti-TGF-β Ab selectively inhibits chronic fibrotic lesions without affecting autoantibody production and the inflammatory component of tissue injury. Thus, TGF-β plays a dual, seemingly paradoxical, role in the development of organ damage in multiorgan autoimmune diseases. According to our working model, reduced TGF-β in immune cells predisposes to immune dysregulation and autoantibody production, which causes tissue inflammation that triggers the production of anti-inflammatory cytokines such as TGF-β in target organs to counter inflammation. Enhanced TGF-β in target organs, in turn, can lead to dysregulated tissue repair, progressive fibrogenesis, and eventual end-organ damage.

Transforming growth factor beta1 (TGFbeta1), an important pleiotropic, immunoregulatory cytokine, uses distinct signaling mechanisms in lymphocytes to affect T-cell homeostasis, regulatory T (Treg)-cell and effector-cell function and tumorigenesis. Defects in TGFbeta1 expression or its signaling in T cells correlate with the onset of several autoimmune diseases. TGFbeta1 prevents abnormal T-cell activation through the modulation of Ca2+-calcineurin signaling in a Caenorhabditis elegans Sma and Drosophila Mad proteins (SMAD)3 and SMAD4-independent manner; however, in Treg cells, its effects are mediated, at least in part, through SMAD signaling. TGFbeta1 also acts as a pro-inflammatory cytokine and induces interleukin (IL)-17-producing pathogenic T-helper cells (Th IL-17 cells) synergistically during an inflammatory response in which IL-6 is produced. Here, we will review TGFbeta1 and its signaling in T cells with an emphasis on the regulatory arm of immune tolerance.

TGFβ1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1−/− CD4+CD8+ and CD4+CD8− thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1−/− thymocytes, whereas the inhibitors of Ca2+ influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1−/− thymocytes is due to a lowered threshold for Ca2+-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFβ1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1−/− mice. Our results suggest for the first time that TGFβ1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca2+-independent stimulatory pathways alone. Consequently, TGFβ1 prevents autoimmune disease through a Ca2+ regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.

Breast cancer is the leading cause of death among female cancer patients. Human epidermal growth factor receptor 2 (HER-2) overexpression is associated with poor prognosis in 15–20% of breast cancer patients. Anti-HER-2 antibody (Herceptin) prolongs the survival of the HER-2+ breast cancer patients. However, resistance to Herceptin is a cause for relapse of aggressive metastatic cancer in these patients. Our earlier studies have demonstrated that PD-L1 blockade enhances the efficacy of HER-2+ breast cancer whole cell vaccine by increasing infiltration of T cells into the tumor. The goal of the present study is to determine the duration of protective anti-tumor memory response to HER-2+ breast cancer. Here, we demonstrate the beneficial effect of GPI-anchored cytokine molecules as adjuvants for generating long lasting memory response against HER-2+ as well as HER-2 negative breast cancer in syngeneic tumor models. Female BALB/c mice were challenged with D2F2/E2 (HER-2 transfected D2F2) cells or D2F2/E2 transfected with GPI-IL-12 or GPI-GM-CSF. While the wild-type challenged mice developed tumors, the mice challenged with transfected D2F2/E2 cells were protected. Protected mice were re-challenged with D2F2/E2 cells 3 months later and D2F2 cells 4 months later. All the mice challenged with D2F2 or D2F2/E2 were protected. We have observed strong antibody response against HER-2 and D2F2 cells in these mice. Our results show that long lasting protective anti-tumor memory response against D2F2 and D2F2/E2 is generated by vaccination with D2F2/E2 cells expressing GPI-anchored immunostimulatory molecules.

Objective The 4T1 triple negative breast cancer (TNBC) model resembles many of the hallmarks of advanced TNBC in humans. In this study we investigate if combining a membrane-based immunotherapy with immune checkpoint blockade antibodies in a metastatic tumor model could effectively generate protective immunity. This personalized strategy is applicable to indications such as TNBC that exhibit a high degree of heterogeneity and do not respond to approved cancer therapies. Methods 4T1 tumor tissue was harvested from BALB/c mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then incorporated with GPI-B7-1 and GPI-IL-12 by protein transfer and used for immunization in conjunction with immune checkpoint inhibitors. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis was assessed by clonogenic assay. Immune response was assessed by IFN-γ ELISPOT. Cell depletion studies were performed with anti-CD4 and anti-CD8 antibodies. Significance was determined using a student’s t test to compare metastasis and immune responses. Results TMV-based immunotherapy in combination with anti-CTLA-4 antibody significantly improved survival, reduced pulmonary metastasis, and increased tumor-specific CD8 T cell responses. Interestingly, either treatment alone failed to have a significant impact on survival, metastasis, or immune response. Conclusions These results strongly suggest that a TMV-based immunotherapy in combination with anti-CTLA-4 antibody generates effective antitumor immunity. Such a combinatorial approach could potentially translate into an effective treatment for patients with metastatic TNBC.

The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGF b receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3−/− mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3−/− mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood. genesis 50:59-66, 2012.

Transforming growth factor beta1 (TGFbeta1) is a multifunctional growth factor involved in wound healing, tissue fibrosis, and in the pathogenesis of many syndromic diseases (e.g., Marfan syndrome, Camurati-Engelmann disease) and muscular, neurological, ophthalmic, cardiovascular and immunological disorders, and cancer. Since the generation of Tgfb1 knockout mice, there has been extraordinary progress in understanding its physiological and pathophysiological function. Here, we report the generation of a conditional knockout allele for Tgfb1 in which its exon 6 is flanked with LoxP sites. As proof of principle, we crossed these mice to LckCre transgenic mice and specifically disrupted Tgfb1 in T cells. The results indicate that T-cell-produced TGFbeta1 is required for normal in vivo regulation of peripheral T-cell activation, maintenance of T-cell homeostasis, and suppression of autoimmunity.

Transforming growth factor β1 (TGFβ1) is an important pleiotropic immunoregulatory cytokine, which is involved in several cellular processes, such as inhibition of T-cell activation, proliferation, differentiation, apoptosis, cytokine production, and effector function. In contrast to its inhibitory effects on naive T cells, it is also involved in stimulation of suppressor/regulatory T-cell expansion and function. In order to achieve so many diverse effects TGFβ utilizes distinct signaling mechanisms in the same cell type. Mouse models with genetic ablation of functional TGFβ signaling mediators have revealed several interesting mechanisms that play an important role in T-cell homeostasis, Treg-cell function, and tumorigenesis. TGFβ1 primarily functions to prevent abnormal T-cell activation through modulation of a Ca2+-calcineurin signaling pathway in a SMAD3/4-independent manner. Additionally, in Treg cells TGFβ1 mediates its effects through a SMAD signaling cascade in a unique manner. TGFβ1 plays duel roles in tumor development and progression.

Transforming growth factor (TGF)β1 is an important immunoregulatory cytokine involved in maintenance of self tolerance and T cell homeostasis [1]. It is produced by several immune and non-immune cell types and functions in both autocrine and paracrine manners [1]–[3]. Regulatory T cells (Treg) cells are the primary source of TGFβ1 for controlling autoimmune disease. Blockade of TGFβ signaling in T cells causes severe autoimmune disease in mice [4, 5]. However, TGFβ signaling in T cells under inflammatory conditions also causes the development of experimentally induced autoimmune encephalomyelitis (EAE), a mouse model for human multiple sclerosis (MS) disease [6]. This review focuses on recent findings concerning the function of TGFβ1 in T cells and immune tolerance, and it discusses implications of these findings for therapeutic intervention in autoimmune and inflammatory diseases.

Optical coherence tomography (OCT) is a high-resolution, nondestructive imaging modality that enables time-serial assessment of adenoma development in the mouse model of colorectal cancer. In this study, OCT was utilized to evaluate the effectiveness of interventions with the experimental antitumor agent α-difluoromethylornithine (DFMO) and a nonsteroidal anti-inflammatory drug sulindac during early [chemoprevention (CP)] and late stages [chemotherapy (CT)] of colon tumorigenesis. Biological endpoints for drug interventions included OCT-generated tumor number and tumor burden. Immunochistochemistry was used to evaluate biochemical endpoints [Ki-67, cleaved caspase-3, cyclooxygenase (COX)-2, β-catenin]. K-Ras codon 12 mutations were studied with polymerase chain reaction-based technique. We demonstrated that OCT imaging significantly correlated with histological analysis of both tumor number and tumor burden for all experimental groups ( P < 0.0001), but allows more accurate and full characterization of tumor number and burden growth rate because of its time-serial, nondestructive nature. DFMO alone or in combination with sulindac suppressed both the tumor number and tumor burden growth rate in the CP setting because of DFMO-mediated decrease in cell proliferation (Ki-67, P < 0.001) and K-RAS mutations frequency ( P = 0.04). In the CT setting, sulindac alone and DFMO/sulindac combination were effective in reducing tumor number, but not tumor burden growth rate. A decrease in COX-2 staining in DFMO/sulindac CT groups (COX-2, P < 0.01) confirmed the treatment effect. Use of nondestructive OCT enabled repeated, quantitative evaluation of tumor number and burden, allowing changes in these parameters to be measured during CP and as a result of CT. In conclusion, OCT is a robust minimally invasive method for monitoring colorectal cancer disease and effectiveness of therapies in mouse models.

To investigate whether the multifocal inflammatory disease in TGFbeta1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1(-/-) and Tgfb1(-/-) Rag1(-/-) mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1(-/-) DO11.10 mice develop a milder inflammation than do Tgfb1(-/-) mice, and their T cells display a less activated phenotype. The lower level of activation correlates with the expression of hybrid TCR (transgenic TCRbeta and endogenous TCRalpha), which could recognize self-Ag and undergo activation. In the complete absence of self-Ag recognition (Tgfb1(-/-) DO11.10 Rag1(-/-) mice) inflammation and T-cell activation are eliminated, demonstrating that self-Ag recognition is required for the hyper-responsiveness of TGFbeta1-deficient T cells. Thus, TGFbeta1 is required for the prevention of autoimmune disease through its ability to control the activation of autoreactive T cells to self-Ag.

Recent studies by Chatenoud and co-workers suggest that non-mitogenic F(ab′)2 fragments of anti-CD3 antibodies, which cannot bind the Fc receptor, induce a prolonged period of tolerance and prevent diabetes in nonobese diabetic (NOD) mice. Tolerance is established by regulatory T cells through the production of transforming growth factor-β1 (TGF-β1), but the mechanism by which TGF-β1 confers this activity is unclear. Analysis of mice deficient in TGF-β1 suggests that TGF-β1 raises the threshold at which intracellular calcium activates T cells to a level that prevents an autoimmune response.

Transforming growth factor beta1 (TGFbeta1) is a potent negative immunoregulatory molecule. We have previously shown that the autoimmune-mediated weaning-age lethality of Tgfb1-/- mice is reversed upon genetic combination with Scid or Rag null alleles. Here, we show that elimination of T but not B cells is sufficient for the reversal, but elimination of either CD4+ or CD8+ cells is not. Although elimination of B cells does not rescue TGFbeta1-deficient animals from autoimmunity, B cells are hyperresponsive to LPS in the absence of TGFbeta1. TGFbeta1 deficiency leads to activation of CD8+ T cells as suggested by down-modulation of CD8 even in the absence of CD4+ T cells. This study provides evidence that both CD4+ and CD8+ T cells, but not B cells, have the ability to cause inflammation in the absence of TGFbeta1. However, though TGFbeta1-deficient B cells are hyperresponsive to stimulation, alone they are not sufficient to cause inflammation.

TGF-β1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-β1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-β1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1−/− mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-γ and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1−/− T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1−/− T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4+ T cells have elevated intracellular Ca2+ levels. However, upon subsequent stimulation in vitro, increases in Ca2+ levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1−/− splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca2+ levels.

The risk of developing lymphoma is markedly increased after solid organ transplantation. Up to 20% of people who receive a solid organ transplant develop a lymphoma. Calcineurin (CN) A beta knockout (Cnab-/-) mice do not reject allogeneic tumors suggesting an immunosuppressive state, is also a unique model for reverse immunosurveillance. Currently, there is no mouse model for low grade lymphomas based on immune dysregulation. Since we observed a spontaneous activation of T cells, and B-cell lymphomas in CNAβ deficient mice, we hypothesize that CN is involved in T-cell regulation, the lack of regulation causes smoldering inflammation which in turn activates B cells and results in lymphomagenesis. The lymphomas in Cnab-/- mice are positive for B cell surface marker (CD21) but negative for T cell (TCRβ) markers and they are clonal in origin and exhibit malignant (destroying the normal architecture of adjoining area) phenotype. In addition, we do see spontaneous activation of T cells in Cnab-/- mice early in life and continue to accumulate, and B cells later when they are old with mild inflammatory lesions in the gut and liver in Helicobacter-free (clean) colony. Since inflammation is considered to be one of the hallmarks of cancer and also the root cause of cancer, this is a novel mouse model for understanding a slow growing, difficult to cure form of lymphomas.