Dysregulated STAT1-SOCS1 control of JAK2 promotes mammary luminal progenitor cell survival and drives ERα+ tumorigenesis

We previously reported that STAT1 expression is frequently abrogated in human estrogen receptor-α-positive (ERα⁺) breast cancers and mice lacking STAT1 spontaneously develop ERα⁺ mammary tumors. However, the precise mechanism by which STAT1 suppresses mammary gland tumorigenesis has not been fully elucidated. Here we show that STAT1-deficient mammary epithelial cells (MECs) display persistent prolactin receptor (PrlR) signaling, resulting in activation of JAK2, STAT3 and STAT5A/5B, expansion of CD61⁺ luminal progenitor cells and development of ERα⁺ mammary tumors. A failure to upregulate SOCS1, a STAT1-induced inhibitor of JAK2, leads to unopposed oncogenic PrlR signaling in STAT1^−/− MECs. Prophylactic use of a pharmacological JAK2 inhibitor restrains the proportion of luminal progenitors and prevents disease induction. Systemic inhibition of activated JAK2 induces tumor cell death and produces therapeutic regression of pre-existing endocrine-sensitive and refractory mammary tumors. Thus, STAT1 suppresses tumor formation in mammary glands by preventing the natural developmental function of a growth factor signaling pathway from becoming pro-oncogenic. In addition, targeted inhibition of JAK2 may have significant therapeutic potential in controlling ERα⁺ breast cancer in humans.     

Resistance to Oncolytic Myxoma Virus Therapy in Nf1?/?/Trp53?/? Syngeneic Mouse Glioma Models Is Independent of Anti-Viral Type-I Interferon

Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 ^+/− Trp53 ^+/− mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection. To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/β is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.     

Increase in IFNγ?IL-2+ Cells in Recent Human CD4 T Cell Responses to 2009 Pandemic H1N1 Influenza

Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1) induced Th1-like responses biased toward the expression of IFNγ⁺TNFα⁺ CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1) induced more IFNγ⁻IL-2⁺TNFα⁺ T cells, similar to the IFNγ⁻IL-2⁺ non-polarized, primed precursor T cells (Thpp) that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα⁺, IFNγ⁺, and IL-2⁺ cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ⁻IL-2⁺TNFα⁺ CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ⁺TNFα⁺) responses. These IFNγ⁻IL-2⁺TNFα⁺ CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.     

CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy

In Chagas disease, CD8⁺ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8⁺ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8⁺ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8⁺ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8⁺ T-cells segregated into IFNγ⁺ perforin (Pfn)^neg or IFNγ^negPfn⁺ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8⁺Pfn⁺ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8 ^−/− recipients showed that the CD8⁺ cells from infected ifnγ^−/− pfn ^+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8⁺ cells from ifnγ ^+/+ pfn ^−/− donors. Moreover, the reconstitution of naïve cd8 ^−/− mice with CD8⁺ cells from naïve ifnγ ^+/+ pfn ^−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ⁺ cells accumulation, whereas reconstitution with CD8⁺ cells from naïve ifnγ ^−/− pfn ^+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn⁺ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8⁺Pfn⁺ and CD8⁺IFNγ⁺ cells during CCC. CD8⁺IFNγ⁺ cells may exert a beneficial role, whereas CD8⁺Pfn⁺ may play a detrimental role in T. cruzi-elicited heart injury.     

Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα⁺ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα⁺ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα⁻ production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67⁺-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67⁺-pDC precursors, none of these being IFNα⁺ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors.     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

γδ T Cells Confer Protection against Murine Cytomegalovirus (MCMV)

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε^−/− mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27⁺ and CD27⁻ γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag^−/−γc^−/− mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients.     

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

Involvment of Cytosolic and Mitochondrial GSK-3β in Mitochondrial Dysfunction and Neuronal Cell Death of MPTP/MPP+-Treated Neurons

Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson''s disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP⁺), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP⁺-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP⁺ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP⁺ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP⁺ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP⁺-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP⁺-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death.     

Genetic disruption of Abl nuclear import reduces renal apoptosis in a mouse model of cisplatin-induced nephrotoxicity

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-μNLS (μ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Abl^μ/μ mice. When injected with cisplatin, we found similar levels of platinum in the Abl^+/+ and the Abl^μ/μ kidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Abl^μ/μ kidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl^+/+ but not in the Abl^μ/μ kidneys. The residual apoptosis in the Abl^μ/μ mice was not further reduced in the Abl^μ/μ; p53^−/− double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl^+/+ and the Abl^μ/μ kidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis.     

Double Negative (CD3+4?8?) TCRαβ Splenic Cells from Young NOD Mice Provide Long-Lasting Protection against Type 1 Diabetes

Background

Double negative CD3⁺4⁻8⁻ TCRαβ splenic cells (DNCD3) can suppress the immune responses to allo and xenografts, infectious agents, tumors, and some autoimmune disorders. However, little is known about their role in autoimmune diabetes, a disease characterized by the reduction of insulin production subsequent to destruction of pancreatic β-cells by a polyclonal population of self-reactive T-cells. Herein, we analyzed the function and phenotype of DNCD3 splenic cells in young NOD mice predisposed to several autoimmune disorders among which, the human-like autoimmune diabetes.

Methodology/Principal Findings

DNCD3 splenic cells from young NOD mice (1) provided long-lasting protection against diabetes transfer in NOD/Scid immunodeficient mice, (2) proliferated and differentiated in the spleen and pancreas of NOD/Scid mice and pre-diabetic NOD mice into IL-10-secreting T_R-1 like cells in a Th2-like environment, and (3) their anti-diabetogenic phenotype is CD3⁺(CD4⁻CD8⁻)CD28⁺CD69⁺CD25^low Foxp3⁻ iCTLA-4⁻TCRαβ⁺ with a predominant Vβ13 gene usage.

Conclusions/Significance

These findings delineate a new T regulatory component in autoimmune diabetes apart from that of NKT and CD4⁺CD25^high Foxp3⁺T-regulatory cells. DNCD3 splenic cells could be potentially manipulated towards the development of autologous cell therapies in autoimmune diabetes.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

Pharmacologic Blockade of JAK1/JAK2 Reduces GvHD and Preserves the Graft-Versus-Leukemia Effect

We have recently reported that interferon gamma receptor deficient (IFNγR−/−) allogeneic donor T cells result in significantly less graft-versus-host disease (GvHD) than wild-type (WT) T cells, while maintaining an anti-leukemia or graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that IFNγR signaling regulates alloreactive T cell trafficking to GvHD target organs through expression of the chemokine receptor CXCR3 in alloreactive T cells. Since IFNγR signaling is mediated via JAK1/JAK2, we tested the effect of JAK1/JAK2 inhibition on GvHD. While we demonstrated that pharmacologic blockade of JAK1/JAK2 in WT T cells using the JAK1/JAK2 inhibitor, INCB018424 (Ruxolitinib), resulted in a similar effect to IFNγR−/− T cells both in vitro (reduction of CXCR3 expression in T cells) and in vivo (mitigation of GvHD after allo-HSCT), it remains to be determined if in vivo administration of INCB018424 will result in preservation of GvL while reducing GvHD. Here, we report that INCB018424 reduces GvHD and preserves the beneficial GvL effect in two different murine MHC-mismatched allo-HSCT models and using two different murine leukemia models (lymphoid leukemia and myeloid leukemia). In addition, prolonged administration of INCB018424 further improves survival after allo-HSCT and is superior to other JAK1/JAK2 inhibitors, such as TG101348 or AZD1480. These data suggest that pharmacologic inhibition of JAK1/JAK2 might be a promising therapeutic approach to achieve the beneficial anti-leukemia effect and overcome HLA-barriers in allo-HSCT. It might also be exploited in other diseases besides GvHD, such as organ transplant rejection, chronic inflammatory diseases and autoimmune diseases.     

Genetic deletion of caspase-2 accelerates MMTV/c-neu-driven mammary carcinogenesis in mice

Despite being the most evolutionarily conserved of the mammalian caspases, little is understood about the cellular function of caspase-2 in normal tissues or what role caspase-2 may have in the progression of human disease. It has been reported that deletion of the caspase-2 gene (Casp2), accelerates Eμ-myc lymphomagenesis in mice, and thus caspase-2 may act as a tumor suppressor in hematological malignancies. Here, we sought to extend these findings to epithelial cancers by examining the potential role of caspase-2 as a tumor suppressor in the mouse mammary carcinogenesis model; MMTV/c-neu. The rate of tumor acquisition was significantly higher in multiparous Casp2^−/−/MMTV mice compared with Casp2^+/+/MMTV and Casp2^+/−/MMTV mice. Cells from Casp2^−/−/MMTV tumors were often multinucleated and displayed bizarre mitoses and karyomegaly, while cells from Casp2^+/+/MMTV and Casp2^+/−/MMTV tumors never displayed this phenotype. Tumors from Casp2^−/−/MMTV animals had a significantly higher mitotic index than tumors from Casp2^+/+/MMTV and Casp2^+/−/MMTV animals. Cell cycle analysis of Casp2^−/− E1A/Ras-transformed mouse embryonic fibroblasts (MEF) also indicated a higher proliferative rate in the absence of caspase-2. In vitro assays further illustrated that MEF had increased genomic instability in the absence of caspase-2. This appears to be due to disruption of the p53 pathway because we observed a concomitant decrease in the induction of the p53 target genes, Pidd, p21 and Mdm2. Thus caspase-2 may function as a tumor suppressor, in part, through regulation of cell division and genomic stability.     

Development of Functional Human NK Cells in an Immunodeficient Mouse Model with the Ability to Provide Protection against Tumor Challenge

Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2^−/−/γ_c ^−/− and NOD/Scid mice, newborn BALB/c Rag2^−/−/γ_c ^−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56⁺ cells in BALB/c Rag2^−/−/γ_c ^−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2^−/−/γ_c ^−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2^−/−/γ_c ^−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2^−/−/γ_c ^−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2^−/−/γ_c ^−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment.     

The Tumor Suppressor Gene,RASSF1A,Is Essential for Protection against Inflammation -Induced Injury

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a^+/−, Rassf1a^−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a ^IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a^−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a^−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a^−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.