Non-Canonical Activin A Signaling Stimulates Context-Dependent and Cellular-Specific Outcomes in CRC to Promote Tumor Cell Migration and Immune Tolerance.

We have shown that activin A (activin), a TGF-ß superfamily member, has pro-metastatic effects in colorectal cancer (CRC). In lung cancer, activin activates pro-metastatic pathways to enhance tumor cell survival and migration while augmenting CD4+ to CD8+ communications to promote cytotoxicity. Here, we hypothesized that activin exerts cell-specific effects in the tumor microenvironment (TME) of CRC to promote anti-tumoral activity of immune cells and the pro-metastatic behavior of tumor cells in a cell-specific and context-dependent manner. We generated an Smad4 epithelial cell specific knockout (Smad4-/-) which was crossed with TS4-Cre mice to identify SMAD-specific changes in CRC. We also performed IHC and digital spatial profiling (DSP) of tissue microarrays (TMAs) obtained from 1055 stage II and III CRC patients in the QUASAR 2 clinical trial. We transfected the CRC cells to reduce their activin production and injected them into mice with intermittent tumor measurements to determine how cancer-derived activin alters tumor growth in vivo. In vivo, Smad4-/- mice displayed elevated colonic activin and pAKT expression and increased mortality. IHC analysis of the TMA samples revealed increased activin was required for TGF-ß-associated improved outcomes in CRC. DSP analysis identified that activin co-localization in the stroma was coupled with increases in T-cell exhaustion markers, activation markers of antigen presenting cells (APCs), and effectors of the PI3K/AKT pathway. Activin-stimulated PI3K-dependent CRC transwell migration, and the in vivo loss of activin lead to smaller CRC tumors. Taken together, activin is a targetable, highly context-dependent molecule with effects on CRC growth, migration, and TME immune plasticity.

LIGHT enhanced bispecific antibody armed T-cells to treat immunotherapy resistant colon cancer.

Increased tumor infiltrating lymphocytes (TIL) are associated with improved patient responses to immunotherapy. As a result, there is interest in enhancing lymphocyte trafficking particularly to colon cancers since the majority are checkpoint blockade-resistant and microsatellite stable. Here, we demonstrate that activated T-cells (ATC) armed with anti-CD3 x anti-EGFR bispecific antibody increases TIL and mediate anti-tumor cytotoxicity while decreasing tumor cell viability. Furthermore, treatment induces endogenous anti-tumor immunity that resisted tumor rechallenge and increased memory T-cell subsets in the tumor. When combined with targeted tumor expression of the tumor necrosis factor superfamily member LIGHT, activated T-cell proliferation and infiltration were further enhanced, and human colorectal tumor regressions were observed. Our data indicate that tumor-targeted armed bispecific antibody increases TIL trafficking and is a potentially potent strategy that can be paired with combination immunotherapy to battle microsatellite stable colon cancer. SIGNIFICANCE: Enhancing trafficking of tumor infiltrating lymphocytes (TILs) to solid tumors has been shown to improve outcomes. Unfortunately, few strategies have been successful in the clinical setting for solid tumors, particularly for "cold" microsatellite stable colon cancers. In order to address this gap in knowledge, this study combined TNFSF14/LIGHT immunomodulation with a bispecific antibody armed with activated T-cells targeted to the tumor. This unique T-cell trafficking strategy successfully generated anti-tumor immunity in a microsatellite stable colon cancer model, stimulated T-cell infiltration, and holds promise as a combination immunotherapy for treating advanced and metastatic colorectal cancer.

Combination Immunotherapy With LIGHT and Interleukin-2 Increases CD8 Central Memory T-Cells In Vivo.

BACKGROUND: The generation of long-term durable tumor immunity and prolonged disease-free survival depends on the ability to generate and support CD8+ central memory T-cells. Microsatellite-stable colon cancer is resistant to currently available immunotherapies; thus, development of novel mechanisms to increase both lymphocyte infiltration and central memory formation are needed to improve outcomes in these patients. We have previously demonstrated that both interleukin-2 (IL-2) and LIGHT (TNFSF14) independently enhance antitumor immune responses and hypothesize that combination immunotherapy may increase the CD8+ central memory T-cell response. METHODS: Murine colorectal cancer tumors were established in syngeneic mice. Tumors were treated with control, soluble, or liposomal IL-2 at established intervals. A subset of animal tumors overexpressed tumor necrosis superfamily factor LIGHT (TNFSF14). Peripheral blood, splenic, and tumor-infiltrating lymphocytes were isolated for phenotypic studies and flow cytometry. RESULTS: Tumors exposed to a combination of LIGHT and IL-2 experienced a decrease in tumor size compared with IL-2 alone that was not demonstrated in wild-type tumors or between other treatment groups. Combination exposure also increased splenic central memory CD8+ cells compared with IL-2 administration alone, while not increasing tumor-infiltrating lymphocytes. In the periphery, the combination enhanced levels of circulating CD8 T-cells and central memory T-cells, while also increasing circulating T-regulatory cells. CONCLUSIONS: Combination of IL-2, whether soluble or liposomal, with exposure to LIGHT results in increased CD8+ central memory cells in the spleen and periphery. New combination immunotherapy strategies that support both effector and memory T-cell functions are critical to enhancing durable antitumor responses and warrant further investigation.

MADD silencing enhances anti-tumor activity of TRAIL in anaplastic thyroid cancer.

ATC is an aggressive disease with limited therapeutic options due to drug resistance. TRAIL is an attractive anti-cancer therapy that can trigger apoptosis in a cancer cell-selective manner. However, TRAIL resistance is a major clinical obstacle for its use as a therapeutic drug. Previously, we demonstrated that MADD is a cancer cell pro-survival factor that can modulate TRAIL resistance. However, its role, if any, in overcoming TRAIL resistance in ATC is unknown. First, we characterized ATC cell lines as either TRAIL resistant, TRAIL sensitive or moderately TRAIL sensitive and evaluated MADD expression/cellular localization. We determined the effect of MADD siRNA on cellular growth and investigated its effect on TRAIL treatment. We assessed the effect of combination treatment (MADD siRNA and TRAIL) on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels. The effect of combination treatment on tumor growth was assessed in vivo. We found increased levels of MADD in ATC cells relative to Nthy-ori 3-1. MADD protein localizes in the cytosol (endoplasmic reticulum and Golgi body) and membrane. MADD knockdown resulted in spontaneous cell death that was synergistically enhanced when combined with TRAIL treatment in otherwise resistant ATC cells. Combination treatment resulted in a significant reduction in MMP and enhanced generation of ROS indicating the putative mechanism of action. In an orthotopic mouse model of TRAIL-resistant ATC, treatment with MADD siRNA alone reduced tumor growth that, when combined with TRAIL, resulted in significant tumor regressions. We demonstrated the potential clinical utility of MADD knockdown in sensitizing cells to TRAIL-induced apoptosis in ATC.

Vaccination With Mitoxantrone-Treated Primary Colon Cancer Cells Enhances Tumor-Infiltrating Lymphocytes and Clinical Responses in Colorectal Liver Metastases.

BACKGROUND: Colorectal cancer remains a leading cause of cancer-related mortality worldwide. Metastases to the liver are often present at initial presentation and will form in most patients during their course of disease. We have previously demonstrated that enhanced trafficking and activation of tumor-infiltrating lymphocytes in colorectal liver metastases (CRLM) may improve antitumor immune responses. Thus, development of novel mechanisms to increase lymphocyte infiltration and activation are needed to improve patient outcomes. METHODS: CT26 murine colorectal cancer cells were treated with physiologic levels of the potent inducer of immunogenic cell death mitoxantrone (MTX). An in situ vaccine was created with treated cells in an established model of CRLM. Cells were evaluated by flow cytometry for cell cycle evaluation and calreticulin expression. Splenic and tumor-infiltrating lymphocytes were isolated for phenotypic studies. RESULTS: MTX-treatment of colon cancer cells resulted in a sub-G1 peak, inhibition of G1 cell cycle progression, and increased G2/M cell fractions while simultaneously increasing dynamic exposure of calreticulin on the cell surface (P < 0.05). Vaccination with MTX-treated cells resulted in significant decreases in CRLM formation associated with increased tumor-infiltrating leukocytes that displayed increased expression of the T cell surface activation marker CD69. CONCLUSIONS: Vaccination with MTX-treated primary colon cancer cells enhances tumor-infiltrating lymphocytes and clinical responses in CRLM.

LIGHT Elevation Enhances Immune Eradication of Colon Cancer Metastases.

The majority of patients with colon cancer will develop advanced disease, with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors that could empower antitumor immune responses in this setting remain to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor cell-specific antitumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte-mediated antitumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Furthermore, we demonstrated evidence of durable tumor-specific antitumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy. Cancer Res; 77(8); 1880-91. ©2017 AACR.

Colon cancer cell treatment with rose bengal generates a protective immune response via immunogenic cell death.

Immunotherapeutic approaches to manage patients with advanced gastrointestinal malignancies are desired; however, mechanisms to incite tumor-specific immune responses remain to be elucidated. Rose bengal (RB) is toxic at low concentrations to malignant cells and may induce damage-associated molecular patterns; therefore, we investigated its potential as an immunomodulator in colon cancer. Murine and human colon cancer lines were treated with RB (10% in saline/PV-10) for cell cycle, cell death, and apoptosis assays. Damage-associated molecular patterns were assessed with western blot, ELISA, and flow cytometry. In an immunocompetent murine model of colon cancer, we demonstrate that tumors regress upon RB treatment, and that RB induces cell death in colon cancer cells through G2/M growth arrest and predominantly necrosis. RB-treated colon cancer cells expressed distinct hallmarks of immunogenic cell death (ICD), including enhanced expression of calreticulin and heat-shock protein 90 on the cell surface, a decrease in intracellular ATP, and the release of HMGB1. To confirm the ICD phenotype, we vaccinated immunocompetent animals with syngeneic colon cancer cells treated with RB. RB-treated tumors served as a vaccine against subsequent challenge with the same CT26 colon cancer tumor cells, and vaccination with in vitro RB-treated cells resulted in slower tumor growth following inoculation with colon cancer cells, but not with syngeneic non-CT26 cancer cells, suggesting a specific antitumor immune response. In conclusion, RB serves as an inducer of ICD that contributes to enhanced specific antitumor immunity in colorectal cancer.

Targeting the metabolic pathway of human colon cancer overcomes resistance to TRAIL-induced apoptosis.

Colon cancer is a leading cause of cancer-related mortality for which targeted therapy is needed; however, trials using apoptosis-inducing ligand monotherapy to overcome resistance to apoptosis have not shown clinical responses. Since colon cancer cells selectively uptake and rapidly metabolize glucose, a property utilized for clinical staging, we investigated mechanisms to alter glucose metabolism in order to selectively target the cancer cells and to overcome evasion of apoptosis. We demonstrate TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) resistance in the majority of human colon cancers tested and utilize the glucose analog 2-deoxy-d-glucose to sensitize TRAIL-resistant gastrointestinal adenocarcinoma cells, and not normal gastrointestinal epithelial cells, to TRAIL-induced apoptosis through enhanced death receptor 5 expression, downstream modulation of MAPK signaling and subsequent miRNA expression modulation by increasing the expression of miR-494 via MEK activation. Further, established human colon cancer xenografts treated with this strategy experience anti-tumor responses. These findings in colon adenocarcinoma support further investigation of manipulation of cellular energetics to selectively overcome resistance to apoptosis and to impart tumor regressions in established colon cancer tumors.

Protein Tyrosine Phosphatase SHP-1 Modulates T Cell Responses by Controlling Cbl-b Degradation.

Previously, we demonstrated that CD28 and CTLA-4 signaling control Casitas-B-lineage lymphoma (Cbl)-b protein expression, which is critical for T cell activation and tolerance induction. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b fails to undergo tyrosine phosphorylation upon CD3 stimulation because SHP-1 is recruited to and dephosphorylates Cbl-b, whereas CD28 costimulation abrogates this interaction. In support of this finding, T cells lacking SHP-1 display heightened tyrosine phosphorylation and ubiquitination of Cbl-b upon TCR stimulation, which correlates with decreased levels of Cbl-b protein. The aberrant Th2 phenotype observed in T cell-specific Shp1(-/-) mice is reminiscent of heightened Th2 response in Cblb(-/-) mice. Indeed, overexpressing Cbl-b in T cell-specific Shp1(-/-) T cells not only inhibits heightened Th2 differentiation in vitro, but also Th2 responses and allergic airway inflammation in vivo. Therefore, SHP-1 regulates Cbl-b-mediated T cell responses by controlling its tyrosine phosphorylation and ubiquitination.

A77 1726, the active metabolite of leflunomide, attenuates lupus nephritis by promoting the development of regulatory T cells and inhibiting IL-17-producing double negative T cells.

Lupus nephritis (LN) is a challenging problem that affects 50% of patients with systemic lupus erythematosus (SLE) without effective therapy. Here, we report that A77 1726, the active metabolite of leflunomide, effectively inhibits development of LN and attenuates the generalized autoimmune features. A77 1726 suppresses the expansion of double negative (DN) T cells, and inhibits T and B cell activation. Intriguingly, A77 1726 treatment significantly increases CD4(+)Foxp3(+) regulatory T cells but suppresses potential "pathogenic" IL-17-producing DN T cells in lymph nodes. In vitro experiment shows that A77 1726 potentiates the conversion of naive CD4(+)CD25(-) T cells into CD4(+)CD25(+)Foxp3(+) inducible regulatory T cells (iTregs) by inhibiting Akt. Taken together, our data indicate that the therapeutic effects of A77 1726 in murine LN are mediated, at least in part, by augmenting iTregs which suppress pathogenic IL-17-producing DN T cells through an Akt-dependent mechanism.

E3 Ubiquitin Ligase Cbl-b Regulates Thymic-Derived CD4+CD25+ Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination.

CD28 costimulation is essential for the development of thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells ("tTregs"). E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. In this paper, we report that the loss of Cbl-b partially but significantly rescues the defective development of tTregs in Cd28(-/-) mice. This partial rescue is independent of IL-2. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in Cd28(-/-) T cells, the defective development of tTregs in Cd28(-/-) mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Stub1 and Cbl-b. Treating Cd28(-/-) mice with a proteasome inhibitor completely rescues defective tTreg development in these mice. Therefore, Cbl-b, together with Stub1, ubiquitinate Foxp3, and regulate tTreg development.

E3 ubiquitin ligase Cbl-b suppresses proallergic T cell development and allergic airway inflammation.

E3 ubiquitin ligase Cbl-b has emerged as a gatekeeper that controls the activation threshold of the T cell antigen receptor and maintains the balance between tolerance and autoimmunity. Here, we report that the loss of Cbl-b facilitates T helper 2 (Th2) and Th9 cell differentiation in vitro. In a mouse model of asthma, the absence of Cbl-b results in severe airway inflammation and stronger Th2 and Th9 responses. Mechanistically, Cbl-b selectively associates with Stat6 upon IL-4 ligation and targets Stat6 for ubiquitination and degradation. These processes are heightened in the presence of T cell receptor (TCR)/CD28 costimulation. Furthermore, we identify K108 and K398 as Stat6 ubiquitination sites. Intriguingly, introducing Stat6 deficiency into Cblb(-/-) mice abrogates hyper-Th2 responses but only partially attenuates Th9 responses. Therefore, our data reveal a function for Cbl-b in the regulation of Th2 and Th9 cell differentiation.

T cell activation threshold regulated by E3 ubiquitin ligase Cbl-b determines fate of inducible regulatory T cells.

E3 ubiquitin ligase Casitas-B-lineage lymphoma protein-b (Cbl-b) is critical for establishing the threshold for T cell activation and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of CD4(+)CD25(+) inducible regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by naive CD4(+)CD25(-) T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is downregulated in vitro. Using TCR-transgenic Rag(-/-) mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.

E3 ubiquitin ligase Cbl-b regulates Pten via Nedd4 in T cells independently of its ubiquitin ligase activity.

E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here, we report that Cbl-b does not inhibit PI3K but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b(-/-) T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b(-/-) mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.

TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha.

Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser(473) in the hydrophobic motif, along with Thr(308) in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr(308), but the kinase(s) responsible for phosphorylating Akt at Ser(473) (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser(473) phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser(473) in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

T-cell receptor-induced NF-kappaB activation is negatively regulated by E3 ubiquitin ligase Cbl-b.

It has previously been shown that E3 ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) negatively regulates T-cell activation, but the molecular mechanism(s) underlying this inhibition is not completely defined. In this study, we report that the loss of Cbl-b selectively results in aberrant activation of NF-kappaB upon T-cell antigen receptor (TCR) ligation, which is mediated by phosphatidylinositol 3-kinase (PI3-K)/Akt and protein kinase C-theta (PKC-theta). TCR-induced hyperactivation of Akt in the absence of Cbl-b may potentiate the formation of caspase recruitment domain-containing membrane-associated guanylate kinase protein 1 (CARMA1)-B-cell lymphoma/leukemia 10 (Bcl10)-mucosa-associated lymphatic tissue 1(MALT1) (CBM) complex, which appears to be independent of PKC-theta. Cbl-b associates with PKC-theta upon TCR stimulation and regulates TCR-induced PKC-theta activation via Vav-1, which couples PKC-theta to PI3-K and allows it to be phosphorylated. PKC-theta then couples IkappaB kinases (IKKs) to the CBM complex, resulting in the activation of the IKK complex. Therefore, our data provide the first evidence to demonstrate that the down-regulation of TCR-induced NF-kappaB activation by Cbl-b is mediated coordinately by both Akt-dependent and PKC-theta-dependent signaling pathways in primary T cells.

Negative regulation of CD40-mediated B cell responses by E3 ubiquitin ligase Casitas-B-lineage lymphoma protein-B.

It has been documented that CD40 is essential for B cell function. Casitas-B-lineage lymphoma protein-b (Cbl-b), an adapter protein and ubiquitin ligase, has been shown to regulate the activation of T and B cells through their Ag receptors. In this study, we report that CD40-induced B cell proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b(-/-) mice display enhanced thymus-dependent Ab responses and germinal center formation, whereas introduction of CD40 deficiency abolishes these effects. Hyper thymus-dependent humoral response in Cbl-b(-/-) mice is in part due to an intrinsic defect in B cells. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-kappaB and JNK. Cbl-b associates with TNF receptor-associated factor 2 upon CD40 ligation, and inhibits the recruitment of TNF receptor-associated factor 2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-kappaB and JNK activation, thereby suppressing B cell responses.

Critical role of vitamin D in sulfate homeostasis: regulation of the sodium-sulfate cotransporter by 1,25-dihydroxyvitamin D3.

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.

[Construction of N-LMP1 transgenic mice with the specific regulation region in nasopharynx].

In order to elucidate the role of EBV-LMP1 in the nasopharyngeal carcinogenesis, the expression vector was constructed with subjecting the N-LMP1 gene to double regulation of two specific regulators: EDL-2 and PLUNC-p. The N-LMP1 related transgenic mice model has been constructed successfully by pronucleus microinjection. 58 founder mice were born, 4 of which were founded to be positive by PCR and Southern blot. Immunohistochemistry assay showed that N-LMP1 protein was expressed in the nasopharynx, tongue and forestomach of transgenic mice.