Human multipotent adult progenitor cells effectively reduce graft-vs-host disease while preserving graft-vs-leukemia activity.

Graft-vs-host disease (GvHD) limits successful outcomes following allogeneic blood and marrow transplantation (allo-BMT). We examined whether the administration of human, bone marrow-derived, multipotent adult progenitor cells (MAPCs™) could regulate experimental GvHD. The immunoregulatory capacity of MAPC cells was evaluated in vivo using established murine GvHD models. Injection of MAPC cells on day +1 (D1) and +4 (D4) significantly reduced T-cell expansion and the numbers of donor-derived, Tumor Necrosis Factor Alpha (TNFα) and Interferon Gamma (IFNγ)-producing, CD4+ and CD8+ cells by D10 compared with untreated controls. These findings were associated with reductions in serum levels of TNFα and IFNγ, intestinal and hepatic inflammation and systemic GvHD as measured by survival and clinical score. Biodistribution studies showed that MAPC cells tracked from the lung and to the liver, spleen, and mesenteric nodes within 24 hours after injection. MAPC cells inhibited mouse T-cell proliferation in vitro and this effect was associated with reduced T-cell activation and inflammatory cytokine secretion and robust increases in the concentrations of Prostaglandin E2 (PGE2) and Transforming Growth Factor Beta (TGFß). Indomethacin and E-prostanoid 2 (EP2) receptor antagonism both reversed while EP2 agonism restored MAPC cell-mediated in vitro T-cell suppression, confirming the role for PGE2. Furthermore, cyclo-oxygenase inhibition following allo-BMT abrogated the protective effects of MAPC cells. Importantly, MAPC cells had no effect on the generation cytotoxic T lymphocyte activity in vitro, and the administration of MAPC cells in the setting of leukemic challenge resulted in superior leukemia-free survival. Collectively, these data provide valuable information regarding the biodistribution and regulatory capacity of MAPC cells, which may inform future clinical trial design.

Cyclin-dependent kinase 5 activity is required for allogeneic T-cell responses after hematopoietic cell transplantation in mice.

Molecular intermediates in T-cell activation pathways are crucial targets for the therapy and prevention of graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (allo-HCT). We recently identified an essential role for cyclin-dependent kinase 5 (Cdk5) in T-cell activation and effector function, but the contribution of Cdk5 activity to the development of GVHD has not been explored. Using an established, preclinical, murine, GVHD model, we reveal that Cdk5 activity is increased in key target organs early after allo-HCT. We then generated chimeric mice (Cdk5+/+C or Cdk5-/-C) using hematopoietic progenitors from either embryonic day 16.5 Cdk5+/+ or Cdk5-/- embryos to enable analyses of the role of Cdk5 in GVHD, as germ line Cdk5 gene deletion is embryonically lethal. The immunophenotype of adult Cdk5-/-C mice is identical to control Cdk5+/+C mice. However, transplantation of donor Cdk5-/-C bone marrow and T cells dramatically reduced the severity of systemic and target organ GVHD. This phenotype is attributed to decreased T-cell migration to secondary lymphoid organs (SLOs), reduced in vivo proliferation within these organs, and fewer cytokine-producing donor T cells during GVHD development. Moreover, these defects in Cdk5-/- T-cell function are associated with altered CCR7 signaling following ligation by CCL19, a receptor:ligand interaction critical for T-cell migration into SLOs. Although Cdk5 activity in donor T cells contributed to graft-versus-tumor effects, pharmacologic inhibition of Cdk5 preserved leukemia-free survival. Collectively, our data implicate Cdk5 in allogeneic T-cell responses after HCT and as an important new target for therapeutic intervention.

Human mesenchymal stromal cells attenuate graft-versus-host disease and maintain graft-versus-leukemia activity following experimental allogeneic bone marrow transplantation.

We sought to define the effects and underlying mechanisms of human, marrow-derived mesenchymal stromal cells (hMSCs) on graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T-cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC-treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T-cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T-cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T-cell proliferation, reduced TNFα, IFNγ, and IL-10 but increased PGE2 levels. Indomethacin and E-prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC-mediated in vitro T-cell suppression, confirming the role for PGE2 . Furthermore, cyclo-oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T-cell proliferation likely through PGE2 induction.

TNF-receptor inhibitor therapy for the treatment of children with idiopathic pneumonia syndrome. A joint Pediatric Blood and Marrow Transplant Consortium and Children's Oncology Group Study (ASCT0521).

Idiopathic pneumonia syndrome (IPS) is an acute, noninfectious lung disorder associated with high morbidity and mortality after hematopoietic cell transplantation. Previous studies have suggested a role for TNFα in the pathogenesis of IPS. We report a multicenter phase II trial investigating a soluble TNF-binding protein, etanercept (Enbrel, Amgen, Thousand Oaks, CA), for the treatment of pediatric patients with IPS. Eligible patients were < 18 years old, within 120 days after transplantation, and with radiographic evidence of a diffuse pneumonitis. All patients underwent a pretherapy broncho-alveolor lavage (BAL) to establish the diagnosis of IPS. Systemic corticosteroids (2.0 mg/kg/day) plus etanercept (.4 mg/kg twice weekly × 8 doses) were administered. Response was defined as survival and discontinuation of supplemental oxygen support by day 28 of study. Thirty-nine patients (median age, 11 years; range, 1 to 17) were enrolled, with 11 of 39 patients nonevaluable because of identification of pathogens from their pretherapy BAL. In the remaining 28 patients, the median fraction of inspired oxygen at study entry was 45%, with 17 of 28 requiring mechanical ventilation. Complete responses were seen in 20 (71%) patients, with a median time to response of 10 days (range, 1 to 24). Response rates were higher for patients not requiring mechanical ventilation at study entry (100% versus 53%, P = .01). Overall survival at 28 days and 1 year after therapy were 89% (95% confidence interval [CI], 70% to 96%) and 63% (95% CI, 42% to 79%), respectively. Plasma levels of proinflammatory cytokines were significantly increased at onset of therapy, subsequently decreasing in responding patients. The addition of etanercept to high-dose corticosteroids was associated with high response rates and survival in children with IPS.

Human biomarker discovery and predictive models for disease progression for idiopathic pneumonia syndrome following allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (SCT) is the only curative therapy for many malignant and nonmalignant conditions. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication that limits successful outcomes. Preclinical models suggest that IPS represents an immune mediated attack on the lung involving elements of both the adaptive and the innate immune system. However, the etiology of IPS in humans is less well understood. To explore the disease pathway and uncover potential biomarkers of disease, we performed two separate label-free, proteomics experiments defining the plasma protein profiles of allogeneic SCT patients with IPS. Samples obtained from SCT recipients without complications served as controls. The initial discovery study, intended to explore the disease pathway in humans, identified a set of 81 IPS-associated proteins. These data revealed similarities between the known IPS pathways in mice and the condition in humans, in particular in the acute phase response. In addition, pattern recognition pathways were judged to be significant as a function of development of IPS, and from this pathway we chose the lipopolysaccaharide-binding protein (LBP) protein as a candidate molecular diagnostic for IPS, and verified its increase as a function of disease using an ELISA assay. In a separately designed study, we identified protein-based classifiers that could predict, at day 0 of SCT, patients who: 1) progress to IPS and 2) respond to cytokine neutralization therapy. Using cross-validation strategies, we built highly predictive classifier models of both disease progression and therapeutic response. In sum, data generated in this report confirm previous clinical and experimental findings, provide new insights into the pathophysiology of IPS, identify potential molecular classifiers of the condition, and uncover a set of markers potentially of interest for patient stratification as a basis for individualized therapy.

Assessment of therapeutic role of mesenchymal stromal cells in mouse models of graft-versus-host disease using cryo-imaging.

Insights regarding the biodistribution and homing of mesenchymal stromal cells (MSCs), as well as their interaction with alloreactive T-cells are critical for understanding how MSCs can regulate graft-versus-host disease (GVHD) following allogeneic (allo) bone marrow transplantation (BMT). We developed novel assays based on 3D, microscopic, cryo-imaging of whole-mouse-sized volumes to assess the therapeutic potential of human MSCs using an established mouse GVHD model. Following infusion, we quantitatively tracked fluorescently labeled, donor-derived, T-cells and third party MSCs in BMT recipients using multispectral cryo-imaging. Specific MSC homing sites were identified in the marginal zones in the spleen and the lymph nodes, where we believe MSC immunomodulation takes place. The number of MSCs found in spleen of the allo BMT recipients was about 200% more than that observed in the syngeneic group. To more carefully define the effects MSCs had on T cell activation and expansion, we developed novel T-cell proliferation assays including secondary lymphoid organ (SLO) enlargement and Carboxyfluoescein succinimidyl ester (CFSE) dilution. As anticipated, significant SLO volume enlargement and CFSE dilution was observed in allo but not syn BMT recipients due to rapid proliferation and expansion of labeled T-cells. MSC treatment markedly attenuated CFSE dilution and volume enlargement of SLO. These assays confirm evidence of potent, in vivo, immunomodulatory properties of MSC following allo BMT. Our innovative platform includes novel methods for tracking cells of interest as well as assessing therapeutic function of MSCs during GVHD induction. Our results support the use of MSCs treatment or prevention of GVHD and illuminate the wider adoption of MSCs as a standard medicinal cell therapy.

Oral TGF-ßR1 inhibitor Vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity.

Osteosarcoma (OS) is an aggressive malignant bone cancer, with refractory and metastatic disease remaining a significant challenge. Transforming growth factor-ß1 (TGF-ß) is a potent immune suppressive cytokine in OS and the TGF-ß is increased in the sera of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-ß1 signaling may be a novel therapy for OS treatment. Here we show that blocking TGF-ß1 signaling using TGF-ßR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells. Vactosertib increased immune effectors (IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (M2-like TAM, MDSC) in the OS tumor microenvironment. Our results suggest that inhibition of TGF-ß1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response.

Functional tumor cell-intrinsic STING, not host STING, drives local and systemic antitumor immunity and therapy efficacy following cryoablation.

BACKGROUND: Despite its potential utility in delivering direct tumor killing and in situ whole-cell tumor vaccination, tumor cryoablation produces highly variable and unpredictable clinical response, limiting its clinical utility. The mechanism(s) driving cryoablation-induced local antitumor immunity and the associated abscopal effect is not well understood. METHODS: The aim of this study was to identify and explore a mechanism of action by which cryoablation enhances the therapeutic efficacy in metastatic tumor models. We used the subcutaneous mouse model of the rhabdomyosarcoma (RMS) cell lines RMS 76-9STINGwt or RMS 76-9STING-/-, along with other murine tumor models, in C57BL/6 or STING-/- (TMEM173-/- ) mice to evaluate local tumor changes, lung metastasis, abscopal effect on distant tumors, and immune cell dynamics in the tumor microenvironment (TME). RESULTS: The results show that cryoablation efficacy is dependent on both adaptive immunity and the STING signaling pathway. Contrary to current literature dictating an essential role of host-derived STING activation as a driver of antitumor immunity in vivo, we show that local tumor control, lung metastasis, and the abscopal effect on distant tumor are all critically dependent on a functioning tumor cell-intrinsic STING signaling pathway, which induces inflammatory chemokine and cytokine responses in the cryoablated TME. This reliance extends beyond cryoablation to include intratumoral STING agonist therapy. Additionally, surveys of gene expression databases and tissue microarrays of clinical tumor samples revealed a wide spectrum of expressions among STING-related signaling components. CONCLUSIONS: Tumor cell-intrinsic STING pathway is a critical component underlying the effectiveness of cryoablation and suggests that expression of STING-related signaling components may serve as a potential therapy response biomarker. Our data also highlight an urgent need to further characterize tumor cell-intrinsic STING pathways and the associated downstream inflammatory response evoked by cryoablation and other STING-dependent therapy approaches.

Cryo-imaging of Stem Cell Biodistribution in Mouse Model of Graft-Versus-Host-Disease.

We demonstrated the use of multispectral cryo-imaging and software to analyze human mesenchymal stromal cells (hMSCs) biodistribution in mouse models of graft-versus-host-disease (GVHD) following allogeneic bone marrow transplantation (BMT). We injected quantum dot labeled MSCs via tail vein to mice receiving BMT and analyzed hMSC biodistribution in major organs (e.g. lung, liver, spleen, kidneys and bone marrow). We compared the biodistribution of hMSCs in mice following allogeneic BMT recipients (with GVHD) to the biodistribution following syngeneic BMT (without GVHD). Cryo-imaging system revealed cellular biodistribution and redistribution patterns in the animal model. We initially found clusters of cells in the lung that eventually dissociated to single cells and redistributed to other organs within 72 h. The in vivo half-life of the exogenous MSCs was about 21 h. We found that the biodistribution of stromal cells was not related to blood flow, rather cells preferentially homed to specific organs. In conclusion, cryo-imaging was suitable for analyzing the cellular biodistribution. It could provide capabilities of visualizing cells anywhere in the mouse model with single cell sensitivity. By characterizing the biodistribution and anatomical specificity of a therapeutic cellular product, we believe that cryo-imaging can play an important role in the advancement of stem and stromal cell therapies and regenerative medicine.

Etanercept clearance during an in vitro model of continuous venovenous hemofiltration.

BACKGROUND/AIMS: Etanercept is a tumor necrosis factor-alpha antagonist used in inflammation-mediated conditions. Continuous venovenous hemofiltration (CVVH) has also been used in patients with inflammatory conditions. This study evaluated etanercept clearance using an in vitro CVVH model. METHODS: Etanercept clearance was assessed in vitro in bovine blood at 1-3 mg/l final serum concentration, and urea control at 750 mg/l. CVVH was performed using polyacrylonitrile, polysulfone, and polymethylmethacrylate filters at 3 l/h ultrafiltrate and 200 ml/min blood flow rates. Transmembrane clearance was estimated using sieving coefficient calculations, and adsorptive removal rate was approximated using a mass balance calculation. RESULTS: Urea sieving coefficient remained constant (1.04 +/- 0.01). Ultrafiltrate etanercept concentrations were undetectable (sieving coefficient < 0.02) and transmembrane and adsorptive clearances were negligible. CONCLUSION: Etanercept is not cleared appreciably by transmembrane or adsorptive mechanisms in CVVH using polyacrylonitrile, polysulfone, or polymethylmethacrylate hemofilters.

Posttransplant Intramuscular Injection of PLX-R18 Mesenchymal-Like Adherent Stromal Cells Improves Human Hematopoietic Engraftment in A Murine Transplant Model.

Late-term complications of hematopoietic cell transplantation (HCT) are numerous and include incomplete engraftment. One possible mechanism of incomplete engraftment after HCT is cytokine-mediated suppression or dysfunction of the bone marrow microenvironment. Mesenchymal stromal cells (MSCs) elaborate cytokines that nurture or stimulate the marrow microenvironment by several mechanisms. We hypothesize that the administration of exogenous MSCs may modulate the bone marrow milieu and improve peripheral blood count recovery in the setting of incomplete engraftment. In the current study, we demonstrated that posttransplant intramuscular administration of human placental derived mesenchymal-like adherent stromal cells [PLacental eXpanded (PLX)-R18] harvested from a three-dimensional in vitro culture system improved posttransplant engraftment of human immune compartment in an immune-deficient murine transplantation model. As measured by the percentage of CD45+ cell recovery, we observed improvement in the peripheral blood counts at weeks 6 (8.4 vs. 24.1%, p < 0.001) and 8 (7.3 vs. 13.1%, p < 0.05) and in the bone marrow at week 8 (28 vs. 40.0%, p < 0.01) in the PLX-R18 cohort. As measured by percentage of CD19+ cell recovery, there was improvement at weeks 6 (12.6 vs. 3.8%) and 8 (10.1 vs. 4.1%). These results suggest that PLX-R18 may have a therapeutic role in improving incomplete engraftment after HCT.

Adoptive natural killer cell therapy is effective in reducing pulmonary metastasis of Ewing sarcoma.

The survival of patients with metastatic or relapsed Ewing sarcoma (ES) remains dismal despite intensification of combination chemotherapy and radiotherapy, precipitating the need for novel alternative therapies with minimal side effects. Natural killer (NK) cells are promising additions to the field of cellular immunotherapy. Adoptive NK cell therapy has shown encouraging results in hematological malignancies. Despite these initial promising successes, however, NK cell therapy for solid tumors remains to be investigated using in vivo tumor models. The purpose of this study is to evaluate the efficacy of ex vivo expanded human NK cells in controlling primary and metastatic ES tumor growth in vitro and in vivo. Using membrane-bound IL-21 containing K562 (K562-mbIL-21) expansion platform, we were able to obtain sufficient numbers of expanded NK (eNK) cells that display favorable activation phenotypes and inflammatory cytokine secretion, along with a strong in vitro cytotoxic effect against ES. Furthermore, eNK therapy significantly decreased lung metastasis without any significant therapeutic effect in limiting primary tumor growth in an in vivo xenograft model. Our data demonstrate that eNK may be effective against pulmonary metastatic ES, but challenges remain to direct proper trafficking and augmenting the cytotoxic function of eNK to target primary tumor sites.

CCL3 Enhances Antitumor Immune Priming in the Lymph Node via IFNγ with Dependency on Natural Killer Cells.

Lymph node (LN) plays a critical role in tumor cell survival outside of the primary tumor sites and dictates overall clinical response in many tumor types (1, 2). Previously, we and others have demonstrated that CCL3 plays an essential role in orchestrating T cell-antigen-presenting cell (APC) encounters in the draining LN following vaccination, and such interactions enhance the magnitude of the memory T cell pool (3-5). In the current study, we investigate the cellular responses in the tumor-draining lymph nodes (TDLNs) of a CCL3-secreting CT26 colon tumor (L3TU) as compared to wild-type tumor (WTTU) during the priming phase of an antitumor response (≤10 days). In comparison to WTTU, inoculation of L3TU resulted in suppressed tumor growth, a phenomenon that is accompanied by altered in vivo inflammatory responses on several fronts. Autologous tumor-derived CCL3 (aCCL3) secretion by L3TU bolstered the recruitment of T- and B-lymphocytes, tissue-migratory CD103+ dendritic cells (DCs), and CD49b+ natural killer (NK) cells, resulting in significant increases in the differentiation and activation of multiple Interferon-gamma (IFNγ)-producing leukocytes in the TDLN. During this early phase of immune priming, NK cells constitute the major producers of IFNγ in the TDLN. CCL3 also enhances CD8+ T cell proliferation and differentiation by augmenting DC capacity to drive T cell activation in the TDLN. Our results revealed that CCL3-dependent IFNγ production and CCL3-induced DC maturation drive the priming of effective antitumor immunity in the TDLN.

Intra-osseous Co-transplantation of CD34-selected Umbilical Cord Blood and Mesenchymal Stromal Cells.

Human mesenchymal stromal cells (MSC) have been shown to support the growth and differentiation of hematopoietic stem cells (HSC). We hypothesized that intra-osseous (IO) co-transplantation of MSC and umbilical cord blood (UCB) may be effective in improving early HSC engraftment, as IO transplantation has been demonstrated to enhance UCB engraftment in NOD SCID-gamma (NSG) mice. Following non-lethal irradiation (300rads), 6 groups of NSG mice were studied: 1) intravenous (IV) UCB CD34+ cells, 2) IV UCB CD34+ cells and MSC, 3) IO UCB CD34+ cells, 4) IO UCB CD34+ cells and IO MSC, 5) IO UCB CD34+ cells and IV MSC, and 6) IV UCB CD34+ and IO MSC. Analysis of human-derived CD45+, CD3+, and CD19+ cells 6 weeks following transplant revealed the highest level of engraftment in the IO UCB plus IO MSC cohort. Bone marrow analysis of human CD13 and CD14 markers revealed no significant difference between cohorts. We observed that IO MSC and UCB co-transplantation led to superior engraftment of CD45+, CD3+ and CD19+ lineage cells in the bone marrow at 6 weeks as compared with the IV UCB cohort controls. Our data suggests that IO co-transplantation of MSC and UCB facilitates human HSC engraftment in NSG mice.

Route of delivery influences biodistribution of human bone marrow-derived mesenchymal stromal cells following experimental bone marrow transplantation.

Mesenchymal stromal cells (MSCs) have shown promise as treatment for graft-versus-host disease (GvHD) following allogeneic bone marrow transplantation (alloBMT). Mechanisms mediating in vivo effects of MSCs remain largely unknown, including their biodistribution following infusion. To this end, human bone-marrow derived MSCs (hMSCs) were injected via carotid artery (IA) or tail vein (TV) into allogeneic and syngeneic BMT recipient mice. Following xenogeneic transplantation, MSC biodistribution was measured by bioluminescence imaging (BLI) using hMSCs transduced with a reporter gene system containing luciferase and by scintigraphic imaging using hMSCs labeled with [(99m)Tc]-HMPAO. Although hMSCs initially accumulated in the lungs in both transplant groups, more cells migrated to organs in alloBMT recipient as measured by in vivo BLI and scintigraphy and confirmed by ex vivo BLI imaging, immunohistochemistry and quantitative RT-PCR. IA injection resulted in persistent whole-body hMSC distribution in alloBMT recipients, while hMSCs were rapidly cleared in the syngeneic animals within one week. In contrast, TV-injected hMSCs were mainly seen in the lungs with fewer cells traveling to other organs. Summarily, these results demonstrate the potential use of IA injection to alter hMSC biodistribution in order to more effectively deliver hMSCs to targeted tissues and microenvironments.

Dysregulated intrahepatic CD4+ T-cell activation drives liver inflammation in ileitis-prone SAMP1/YitFc mice.

BACKGROUND AND AIMS: Liver inflammation is a common extraintestinal manifestation of inflammatory bowel disease (IBD); however, whether liver involvement is a consequence of a primary intestinal defect or results from alternative pathogenic processes remains unclear. Therefore, we sought to determine the potential pathogenic mechanism(s) of concomitant liver inflammation in an established murine model of IBD. METHODS: Liver inflammation and immune cell subsets were characterized in ileitis-prone SAMP1/YitFc (SAMP) and AKR/J (AKR) control mice, lymphocyte-depleted SAMP (SAMPxRag-1-/-), and immunodeficient SCID recipient mice receiving SAMP or AKR donor CD4+ T-cells. Proliferation and suppressive capacity of CD4+ T-effector (Teff) and T-regulatory (Treg) cells from gut-associated lymphoid tissue (GALT) and livers of SAMP and AKR mice were measured. RESULTS: Surprisingly, prominent inflammation was detected in 4-wk-old SAMP livers, prior to histologic evidence of ileitis, while both disease phenotypes were absent in age-matched AKRs. SAMP liver disease was characterized by abundant infiltration of lymphocytes, required for hepatic inflammation to occur, a Th1-skewed environment, and phenotypically-activated CD4+ T-cells. SAMP intrahepatic CD4+ T-cells also had the ability to induce liver and ileal inflammation when adoptively transferred into SCID recipients, whereas GALT-derived CD4+ T-cells produced milder ileitis, but not liver inflammation. Interestingly, SAMP intrahepatic CD4+ Teff cells showed increased proliferation compared to both SAMP GALT- and AKR liver-derived CD4+ Teff cells, while SAMP intrahepatic Tregs were decreased among CD4+ T-cells and impaired in in vitro suppressive function compared to AKR. CONCLUSIONS: Activated intrahepatic CD4+ T-cells induce liver inflammation and contribute to experimental ileitis via locally-impaired hepatic immunosuppressive function.

Murine leukemia virus envelope gp70 is a shared biomarker for the high-sensitivity quantification of murine tumor burden.

The preclinical development of anticancer drugs including immunotherapeutics and targeted agents relies on the ability to detect minimal residual tumor burden as a measure of therapeutic efficacy. Real-time quantitative (qPCR) represents an exquisitely sensitive method to perform such an assessment. However, qPCR-based applications are limited by the availability of a genetic defect associated with each tumor model under investigation. Here, we describe an off-the-shelf qPCR-based approach to detect a broad array of commonly used preclinical murine tumor models. In particular, we report that the mRNA coding for the envelope glycoprotein 70 (gp70) encoded by the endogenous murine leukemia virus (MuLV) is universally expressed in 22 murine cancer cell lines of disparate histological origin but is silent in 20 out of 22 normal mouse tissues. Further, we detected the presence of as few as 100 tumor cells in whole lung extracts using qPCR specific for gp70, supporting the notion that this detection approach has a higher sensitivity as compared with traditional tissue histology methods. Although gp70 is expressed in a wide variety of tumor cell lines, it was absent in inflamed tissues, non-transformed cell lines, or pre-cancerous lesions. Having a high-sensitivity biomarker for the detection of a wide range of murine tumor cells that does not require additional genetic manipulations or the knowledge of specific genetic alterations present in a given neoplasm represents a unique experimental tool for investigating metastasis, assessing antitumor therapeutic interventions, and further determining tumor recurrence or minimal residual disease.