Mouse T-cell Research Articles

Leptin promotes lupus T-cell autoimmunity

In systemic lupus erythematosus (SLE), the impairment in apoptosis can facilitate the initiation and maintenance of autoimmune responses to self antigens. Here we show that the adipocytokine leptin, which is abnormally elevated in SLE, promotes the survival and proliferation of autoreactive T-cells in mice with an autoreactive T-cell repertoire, including (NZB x NZW)F1 lupus-prone mice. This ability of leptin to promote lupus T-cell autoimmunity suggests the possibility of a therapeutic targeting of leptin in SLE.

Learning local directed acyclic graphs based on multivariate time series data

Multivariate time series (MTS) data such as time course gene expression data in genomics are often collected to study the dynamic nature of the systems. These data provide important information about the causal dependency among a set of random variables. In this paper, we introduce a computationally efficient algorithm to learn directed acyclic graphs (DAGs) based on MTS data, focusing on learning the local structure of a given target variable. Our algorithm is based on learning all parents (P), all children (C) and some descendants (D) (PCD) iteratively, utilizing the time order of the variables to orient the edges. This time series PCD-PCD algorithm (tsPCD-PCD) extends the previous PCD-PCD algorithm to dependent observations and utilizes composite likelihood ratio tests (CLRTs) for testing the conditional independence. We present the asymptotic distribution of the CLRT statistic and show that the tsPCD-PCD is guaranteed to recover the true DAG structure when the faithfulness condition holds and the tests correctly reject the null hypotheses. Simulation studies show that the CLRTs are valid and perform well even when the sample sizes are small. In addition, the tsPCD-PCD algorithm outperforms the PCD-PCD algorithm in recovering the local graph structures. We illustrate the algorithm by analyzing a time course gene expression data related to mouse T-cell activation.

Th1‐mediated experimental autoimmune encephalomyelitis is CXCR3 independent

Drugs that block leukocyte trafficking ameliorate multiple sclerosis (MS). Occurrences of opportunistic infection, however, highlight the need for novel drugs that modulate more restricted subsets of T cells. In this context, chemokines and their receptors are attractive therapeutic targets. CXCR3, a Th1-associated chemokine receptor, is preferentially expressed on T cells that accumulate in MS lesions and central nervous system (CNS) infiltrates of mice with experimental autoimmune encephalomyelitis (EAE). Surprisingly, mice genetically deficient in either CXCR3 or CXCL10 succumb to EAE following active immunization with myelin antigens. EAE is mediated by a heterogeneous population of Tcells in myelin-immunized mice. Hence, disease might develop in the absence of CXCR3 secondary to the compensatory action of encephalitogenic CCR6(+) Th17 cells. However, in the current study, we show for the first time that blockade or genetic deficiency of either CXCR3 or of its primary ligand has no impact on clinical EAE induced by the adoptive transfer of highly polarized Th1 effector cells. Our data illustrate the fact that, although highly targeted immunotherapies might have more favorable side effect profiles, they are also more likely to be rendered ineffective by inherent redundancies in chemokine and cytokine networks that arise at sites of neuroinflammation.

Characterization and Optimization of the Glucan Particle-Based Vaccine Platform

Glucan particles (GPs) are hollow porous Saccharomyces cerevisiae cell walls that are treated so that they are composed primarily of β-1,3-d-glucans. Our previous studies showed that GPs can serve as an effective vaccine platform. Here, we characterize CD4(+) T-cell and antibody responses in immunized mice as a function of antigen (ovalbumin) encapsulation, antigen dose, particle numbers, time, immunization schedule, and trapping methods. Although we found that GPs served as an effective adjuvant when admixed with free antigens for IgG1 antibody production, stronger CD4(+) T-cell and IgG2c antibody responses were stimulated when antigens were encapsulated inside GPs, suggesting that the GP platform acts as both an adjuvant and a delivery system. Vigorous T-cell and antibody responses were stimulated even at submicrogram antigen doses, as long as the number of GPs was kept at 5 × 10(7) particles per immunization. One prime and one boost were sufficient to elicit robust immune responses. In addition, strong antigen-specific antibody and T-cell responses prevailed up to 20 months following the last immunization, including those of gamma interferon (IFN-γ), interleukin 17A (IL-17A), and dual IFN-γ/IL-17A-secreting CD4(+) T cells. Finally, robust immune responses were observed using generally recognized as safe (GRAS) materials (alginate and calcium, with or without chitosan) to trap antigens within GPs. Thus, these studies demonstrate that antigens encapsulated into GPs make an effective vaccine platform that combines adjuvanticity and antigen delivery to elicit strong durable immune responses at relatively low antigen doses using translationally relevant formulations.

T Cell-Derived Protein S Engages TAM Receptor Signaling in Dendritic Cells to Control the Magnitude of the Immune Response

Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactiveimmune responses. Tcells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse Tcells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to Tcell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human Tcells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.

B7-H4Ig inhibits mouse and human T-cell function and treats EAE via IL-10/Treg-dependent mechanisms

We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4+ T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4+ T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4+ T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.

T-Cell Reconstitution after Thymus Xenotransplantation Induces Hair Depigmentation and Loss

Here we present a mouse model for T-cell targeting of hair follicles, linking the pathogenesis of alopecia to that of depigmentation disorders. Clinically, thymus transplantation has been successfully used to treat T-cell immunodeficiency in congenital athymia, but is associated with autoimmunity. We established a mouse model of thymus transplantation by subcutaneously implanting human thymus tissue into athymic C57BL/6 nude mice. These xenografts supported mouse T-cell development. Surprisingly, we did not detect multiorgan autoimmune disease. However, in all transplanted mice, we noted a striking depigmentation and loss of hair follicles. Transfer of T cells from transplanted nudes to syngeneic black-coated RAG−/− recipients caused progressive, persistent coat-hair whitening, which preceded patchy hair loss in depigmented areas. Further transfer experiments revealed that these phenomena could be induced by CD4+ T cells alone. Immunofluorescent analysis suggested that Trp2+ melanocyte-lineage cells were decreased in depigmented hair follicles, and pathogenic T cells upregulated activation markers when exposed to C57BL/6 melanocytes in vitro, suggesting that these T cells are not tolerant to self-melanocyte antigens. Our data raise interesting questions about the mechanisms underlying tissue-specific tolerance to skin antigens.

The Impact of Lactobacillus plantarum WCFS1 Teichoic Acid D-Alanylation on the Generation of Effector and Regulatory T-cells in Healthy Mice

To date it remains unclear how probiotics affect the immune system. Bacterial envelope components may play an essential role, as these are the first to establish bacterial-host cell interactions. Teichoic acids (TAs), and especially lipoteichoic acids, are the most pro-inflammatory components of the gram-positive bacterial envelope. This effect is dependent on D-alanyl substitution of the TA backbone and interactions with TLR2 on host cells. Although the pro-inflammatory properties of TAs have been established in vitro, it remains unclear how TAs affect immunomodulation in vivo. In this study, we investigated the role of TA D-alanylation on L. plantarum–induced intestinal and systemic immunomodulation in vivo. For this, we compared the effect of L. plantarum WCFS1 and its TA D-Alanylation negative derivative (dltX-D) on the distribution of dendritic cell and T cell populations and responses in healthy mice. We demonstrated that the majority of the L. plantarum-induced in vivo immunomodulatory effects were dependent on D-alanylation (D-Ala), as some L. plantarum WCFS1-induced immune changes were not observed in the dltX-D-treated group and some were only observed after treatment with dltX-D. Strikingly, not only pro-inflammatory immune responses were abolished in the absence of D-Ala substitution, but also anti-inflammatory responses, such as the L. plantarum-induced generation of regulatory T cells in the spleen. With this study we provide insight in host-microbe interactions, by demonstrating the involvement of D-alanylation of TAs on the bacterial membrane in intestinal and systemic immunomodulation in healthy mice.

Abstract 4487: Synergistic effects of resveratrol and doxorubicin on inducing the apotosis of mouse and human PTEN deficient T-cell acute lymphoblastic cells.

Abstract Resveratrol (3,5,4’-trihydroxy-trans-stilbene), a plant antibiotic produced in a wide variety of plants in response to stress, injury, UV irradiation, and fungal infection, has been shown to inhibit cell growth of several types of cancer. The aim of the present study was to determine the effects of Resveratrol alone and its combination with Doxorubicin, a commonly used chemotherapeutics for T-cell acute lymphoblastic leukemia (T-ALL), on the proliferation and apoptosis of T-ALL cells. Mouse T-ALL cell lines were derived from T-cell-specific deletion of Pten tumor suppressor gene, and human T-ALL cells were obtained from ATCC. We treated T-ALL cells with different concentrations of Resveratrol (0 μM to 200 μM) alone and in combination with various concentrations of Doxorubicin (0 μM to 1 μM) for different durations (0, 12, 24, 48 and 72 hours, respectively). We showed mouse and human Pten deficient T-ALL cell lines were sensitive to the treatment of Resveratrol in dose- and time-dependent manners. In addition, low dose Resveratrol (3.125 μM) in combination with very low dose of o (0.01 μM) showed a remarkable synergistic effect on the cell proliferation and apoptosis. Flow cytometric analysis with Annexin V and Western blot studies on Caspase 3 confirmed that Resveratrol induced apoptosis, especially in combination with Doxorubicin. This study establishes a potential role of Resveratrol in the treatment of Pten deficient T-ALL. Citation Format: Ling Chen, Yaling Yang, M. James You. Synergistic effects of resveratrol and doxorubicin on inducing the apotosis of mouse and human PTEN deficient T-cell acute lymphoblastic cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2013-4487

Abstract 3144: shRNA library screening by dual shRNA technique identifies CHEK1 as a synthetic lethal gene in the absence of IKKε in ovarian cancer.

Abstract Background: IKKε was previously identified as an oncogene in breast cancer and was associated with poor clinical outcome in ovarian cancer. Recently, we demonstrated that IKKε is a key regulator of invasion and metastasis programs in ovarian cancer. Therefore, we developed a rapid and robust dual shRNA technique to create IKKε-matched cell line pairs and performed synthetic lethality screens to identify the key molecular targets influenced by the loss of IKKε expression in ovarian cancer. Material and methods: To create an IKKε-matched cell line pair, we utilized a retroviral bicistronic vector containing the transmembrane and extracellular domains of the mouse T-cell surface glycoprotein CD8 alpha, co-expressed with either IKKε or negative control shRNAs. The positively transduced cells were isolated using mouse CD8a-specific microbeads. After recovery from beads purification, the human kinome shRNA library, containing a puromycin selectable marker fused to GFP, was introduced. To identify shRNAs that sensitized ovarian cancer cells to IKKε depletion, we harvested each shRNA library infected control and IKKε- depleted cells at two time points. This screening was performed in two different ovarian cancer cell lines, one with and one without p53 mutation. Results: We prioritized the genes consistently depleted at two different time points, depending on their doubling times (A2780 at Days 4 and 7, Ovcar5 at Days 7 and 14). In order to generalize this synthetic lethality to ovarian cancers expressing a high level of IKKε, we first focused on the genes identified in both ovarian cancer cell lines, yielding 29 candidate genes. We examined the expression levels of these 29 candidate genes in The Cancer Genome Atlas data portal containing more than 500 ovarian serous cystadenocarcinoma. Most of these 29 genes were overexpressed in a subset of TCGA ovarian cancers. Strikingly, CHEK1 was overexpressed by more than 2 fold in 98% of TCGA ovarian cancers. The combination knockdown with IKKε and CHEK1 resulted in a further decrease in cellular proliferation and a further increase in G0/G1 arrest compared to individual depletion of either gene. Consistently, we observed the same biological effect using pharmacological inhibitors of IKKε (BX795) and CHEK1 (PF00477736). Conclusion: Our dual shRNA technique efficiently identified CHEK1 as an IKKε synthetic lethal gene in ovarian cancer. Identification of IKKε dependent signaling will lead to development of context-specific therapeutics in the poor prognostic group of patients with a high level of IKKε expression. Citation Format: Marianne K H Kim, George Wright, Louis Staudt, Christina Annunziata. shRNA library screening by dual shRNA technique identifies CHEK1 as a synthetic lethal gene in the absence of IKKε in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3144. doi:10.1158/1538-7445.AM2013-3144

Treatment of Metastatic Renal Cell Carcinoma With CAIX CAR-engineered T cells: Clinical Evaluation and Management of On-target Toxicity

Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 10(9) CAR T cells. CTC grade 2-4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 10(9) T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed "on-target" toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses.

Hcp and VgrG1 are secreted components of theHelicobacter hepaticustype VI secretion system and VgrG1 increases the bacterial colitogenic potential

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T-cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS-dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T-cell-transferred C57BL/6 Rag2(-/-) mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild-typeH. hepaticus as compared with mice infected with VgrG1-deficient isogenic bacteria. In addition, competitive assays between both wild-type and T6SS-deficient H. hepaticus, and between wild-type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro-inflammatory potential mediated by the T6SS. Cellular innate pro-inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro-inflammatory effect of the chronic H. hepaticus infection.

Molecular profiling of LFA-1 signalling in T-cells identifies novel genomic signatures implicated in Th1, Th17 and iTreg polarization

Event Abstract Back to Event Molecular profiling of LFA-1 signalling in T-cells identifies novel genomic signatures implicated in Th1, Th17 and iTreg polarization Navin K. Verma1*, Eugene Dempsey1, Sean P. Barry1, Anthony Davies1, Aideen Long1, Padraic Fallon1, 2, Yuri Volkov1 and Dermot Kelleher1, 3 1 Trinity College Dublin, Institute of Molecular Medicine, Ireland 2 Our Lady’s Children’s Hospital, National Children’s Research Centre, Ireland 3 Imperial College London, Faculty of Medicine, United Kingdom Background: T-cell expression of functional phenotypes is dependent on a range of interacting signals. Here we demonstrate molecular mechanisms by which LFA-1-mediated signalling associated with lymphocyte migration modulates T-cell differentiation into Th1, Th17 or iTreg. Methods: Primary human or mouse T-cells were stimulated via LFA-1 by incubating on immobilized recombinant ICAM-1. Affymetrix GeneChip® microarrays and Ingenuity Pathway Analysis were performed. Biochemical and imaging techniques including real-time PCR, Western-blotting, ELISA, siRNA-mediated gene silencing, confocal microscopy and High Content Analysis were utilized. Results: Molecular profiling of LFA-1-stimulated T-cells identified genomic signatures defining both Notch and TGF-β signalling pathways. We further demonstrate that LFA-1/ICAM-1 interaction activates Notch signalling by nuclear translocation of its cleaved intracellular domain and up-regulation of target genes Hey1 and Hes1. This interaction also up-regulates a subset of molecules associated with reduced TGF-β responsiveness, including Smad7, Smurf2 and Ski. The increased expression of these molecules in T-cells significantly attenuates TGF-β-mediated phosphorylation of Smad2 and/or suppression of IL-2 secretion. While LFA-1/ICAM-1 favours Notch-dependent Tbet+ Th1 polarization, LFA-1-stimulated T-cells are refractory to TGF-β-mediated induction of Foxp3+ iTreg or RORγt+ Th17 differentiation. Pre-treatment of T-cells with blocking anti-LFA-1 antibody, specific inhibitors or siRNA against identified genes substantially antagonises LFA-1/ICAM-1-mediated effects on functional phenotypes and restores their TGF-β sensitivity. Conclusion: This study establishes a crucial role of LFA-1-mediated signalling in immunoregulation concurrent with lymphocyte motility, involving both Notch and TGF-β pathways. Our findings suggest a rational basis for novel selective and “tunable” therapeutic approaches aimed to modulate immune functions in inflammatory diseases. Keywords: LFA-1, ICAM-1, Th17, iTreg, Th1 Cells, TGF-beta, Notch signaling Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Verma NK, Dempsey E, Barry SP, Davies A, Long A, Fallon P, Volkov Y and Kelleher D (2013). Molecular profiling of LFA-1 signalling in T-cells identifies novel genomic signatures implicated in Th1, Th17 and iTreg polarization. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00388 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 19 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Navin K Verma, Trinity College Dublin, Institute of Molecular Medicine, Dublin, Ireland, verman@tcd.ie Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Navin K Verma Eugene Dempsey Sean P Barry Anthony Davies Aideen Long Padraic Fallon Yuri Volkov Dermot Kelleher Google Navin K Verma Eugene Dempsey Sean P Barry Anthony Davies Aideen Long Padraic Fallon Yuri Volkov Dermot Kelleher Google Scholar Navin K Verma Eugene Dempsey Sean P Barry Anthony Davies Aideen Long Padraic Fallon Yuri Volkov Dermot Kelleher PubMed Navin K Verma Eugene Dempsey Sean P Barry Anthony Davies Aideen Long Padraic Fallon Yuri Volkov Dermot Kelleher Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ca2+ Signaling in T-Cell Subsets with a Focus on the Role of Cav1 Channels: Possible Implications in Therapeutics

OPINION article Front. Immunol., 20 June 2013Sec. T Cell Biology https://doi.org/10.3389/fimmu.2013.00150

Mesenchymal Stem Cell Lines Isolated by Different Isolation Methods Show Variations in the Regulation of Graft-versus-host Disease

Since the discovery of the immunomodulation property of mesenchymal stem cells (MSCs) about a decade ago, it has been extensively investigated whether MSCs can be used for the treatment of immune-related diseases, such as graft-versus-host disease (GvHD). However, how to evaluate the efficacy of human MSCs for the clinical trial is still unclear. We used an MHC-mismatched model of GvHD (B6 into BALB/c). Surprisingly, the administration of the human MSCs (hMSCs) could reduce the GvHD-related mortality of the mouse recipients and xenogeneically inhibit mouse T-cell proliferation and IFN-γ production in vitro. We recently established a new protocol for the isolation of a homogeneous population of MSCs called subfractionation culturing methods (SCM), and established a library of clonal MSC lines. Therefore, we also investigated whether MSCs isolated by the conventional gradient centrifugation method (GCM) and SCM show different efficacy in vivo. Intriguingly, clonal hMSCs (hcMSCs) isolated by SCM showed better efficacy than hMSCs isolated by GCM. Based on these results, the MHC-mismatched model of GvHD may be useful for evaluating the efficacy of human MSCs before the clinical trial. The results of this study suggest that different MSC lines may show different efficacy in vivo and in vitro.

Targeting Thymic Epithelia AR Enhances T-Cell Reconstitution and Bone Marrow Transplant Grafting Efficacy

Although thymic involution has been linked to the increased testosterone in males after puberty, its detailed mechanism and clinical application related to T-cell reconstitution in bone marrow transplantation (BMT) remain unclear. By performing studies with reciprocal BMT and cell-specific androgen receptor (AR) knockout mice, we found that AR in thymic epithelial cells, but not thymocytes or fibroblasts, played a more critical role to determine thymic cellularity. Further dissecting the mechanism using cell-specific thymic epithelial cell-AR knockout mice bearing T-cell receptor transgene revealed that elevating thymocyte survival was due to the enhancement of positive selection resulting in increased positively selected T-cells in both male and female mice. Targeting AR, instead of androgens, either via genetic knockout of thymic epithelial AR or using an AR-degradation enhancer (ASC-J9®), led to increased BMT grafting efficacy, which may provide a new therapeutic approach to boost T-cell reconstitution in the future.

Immune responses and protective efficacy of a recombinant swinepox virus co-expressing HA1 genes of H3N2 and H1N1 swine influenza virus in mice and pigs

The recombinant swine poxvirus rSPV/H3-2A-H1 co-expressing HA1 genes of H3N2 and H1N1 subtype SIV has been constructed and identified. Inoculations of rSPV/H3-2A-H1 yielded ELISA and neutralization antibodies against SIV H1N1 and H3N2, and elicited potent H1N1 and H3N2 SIV-specific INF-γ response from T-lymphocytes in mice and pigs in this study. Complete protection against SIV H1N1 or H3N2 challenge in pigs was observed.

Vps41, a protein involved in lysosomal trafficking, interacts with caspase-8.

Caspase-8 is a member of the cysteine-aspartic acid protease (caspase) family which plays a central role in apoptosis and development. We screened caspase-8 interacting proteins from mouse T-cell lymphoma and 7.5-day embryo cDNA libraries by yeast two-hybrid system and obtained eleven positive clones, including Vacuolar protein sorting 41 (Vps41), a protein involved in trafficking of proteins from the late Golgi to the vacuole. The interaction of Vps41 with caspase-8 was confirmed by co-immunoprecipitation (co-IP) and co-localization studies in HEK293T cells. Co-IP experiments also showed that Vps41 binds to the p18 subunit of caspase-8 through its WD40 region and RING-finger motif. Furthermore, we found that overexpression of Vps41 promotes Fas-induced apoptosis in A549 human lung adenocarcinoma cells. The cleavage of caspase-3, a caspase-8 downstream effector, was increased when cells were transfected with Vps41-overexpressing plasmid. Together, these results suggest a novel interaction of caspase-8 with Vps41 and provide a potential role of Vps41 beyond lysosomal trafficking.

Discovering Optimal Targets for Adoptive T-Cell Immunotherapy of Leukemia.

AbstractAbstract 3016Allogeneic hematopoietic cell transplantation is the most widely used form of adoptive T-cell cancer immunotherapy (ATCI) but its efficacy is limited by the fact that donor lymphocytes are neither selected nor primed (activated) prior to transfer. This can lead to tolerization of donor lymphocytes by tumor cells and causes graft-versus-host disease in 60% of recipients. These two caveats can be circumvented by the injection of primed antigen-specific CD8 T-cells targeted to tumor associated antigens (TAAs) or minor histocompatibility antigens (MiHAs). Several studies in humans have established the value of TAAs and MiHAs in therapies against solid tumors and leukemia, respectively. Importantly, the value of TAAs and MiHAs as targets for antigen-specific cancer immunotherapy has never been assessed against the same tumor.Therefore, our main goal is to directly compare the therapeutic efficacy of T-cells targeted to TAAs vs. MiHAs. More specifically, we want to evaluate the in vivo anti-leukemic potential of MiHA- vs. TAA-primed CD8 T-cells and identify the mechanisms responsible for the differential anti-leukemic activity of antigen-specific T-cells.We elected to work with 8 antigens known to be expressed on a mouse lymphoblastoma cell line (EL4 cells). Our panel includes 4 MiHAs of known sequence. We also selected 4 TAAs, whose sequences were elucidated by our group using a high-throughput mass spectrometry based approach (Fortier M.H. et al. 2008, J. Exp. Med). In vitro cytotoxicity assays revealed a functional CTL response against all of our antigens, thereby confirming their immunogenicity.We first evaluated the anti-leukemic activity of antigen-specific CD8 T cells in vivo. Mice were immunized twice with peptide pulsed dendritic cells and challenged with 5 × 105 EL4 cells 7 days after the last immunization. We observed an enhanced survival rate for mice immunized against MiHAs as opposed to mice immunized against TAAs. Indeed, while none of the mice immunized against TAAs survived the EL4 challenge, mice immunized against 3 out of the 4 MiHAs demonstrated either full survival (2 MiHAs) or partial survival with a delayed tumor onset.To better understand the mechanisms responsible for the differential anti-leukemic activity of antigen-specific T-cells, we assessed the quality of binding of the 8 antigens to the MHC I molecule. More specifically, we measured the half-life of MHC I/peptide complexes at the cell surface and performed a peptide binding competition assay to evaluate the affinity of our 8 antigens for MHC I molecules. Notably, MiHAs and TAAs showed comparable half-life of MHC I/peptide complexes at the cell surface and binding affinities. We conclude that differential MHC I/peptide interactions are not responsible for the differential anti-leukemic activity of MiHA- vs. TAA-primed CD8 T-cells.We then investigated the frequency of antigen-specific CD8 T-cells in immunized mice using MHC class I tetramers. Our preliminary results show that CD8 T-cells specific for 4 of our antigens (3 TAAs and 1 MiHA) are undetectable by flow cytometry. Interestingly, we found a strong correlation between the lack of tetramer staining and lack of survival of EL4 bearing mice. This suggests that CD8 T-cells targeting TAAs bind weakly to their respective MHC I/peptide complexes, which could indicate a weaker immunogenicity compared to MiHAs. It will be interesting to investigate whether and how other mechanisms of immunogenicity, such as TCR avidity and T-cell frequency, can influence the outcome of antigen-specific leukemia immunotherapy.Our studies will provide the first direct comparison of the anti-leukemic potential of MiHA- vs. TAA-primed CD8 T-cells. We believe that these crucial informations will serve as guide for selecting the best antigens for antigen-specific ATCI in future clinical trials. Disclosures:No relevant conflicts of interest to declare.

Kruppel-Like Factor, Klf9, Promotes Proliferation of Notch-Independent Precursor T-Cell Lymphoblastic Lymphomas

AbstractAbstract 3496Notch1 signaling is required at multiple stages of normal T-lymphocyte development. Notch1 is a transmembrane receptor that is physiologically activated when Notch ligands induce conformational changes that allow Notch1 cleavage by the intramembranous γ-secretase complex, releasing active intracellular Notch1 (ICN1) fragment from the plasma membrane. Activating NOTCH1 mutations are very frequent in human and mouse T-cell lymphoblastic leukemia/lymphoma (T-LL). Typically, these mutations promote ligand-independent Notch1 cleavage by γ-secretase or increase ICN1 stability by truncating the C-terminal PEST domain. Understandably, much effort has focused on elucidating mechanisms of normal and oncogenic Notch1 signaling. However, some studies suggest that the absence of NOTCH1 mutations portends a worse prognosis for human T-LL. Therefore, we set out to define signals that promote proliferation and survival of T-LL cells lacking activated Notch1. We used Western blotting to detect γ-secretase cleaved ICN1 protein in a cohort of 35 primary T-LLs that developed spontaneously in mice lacking the Ataxia telangiectasia mutated (Atm) tumor suppressor. We identified 3 ICN1 subgroups: 63% expressed PEST-truncated ICN1 (T-ICN1); 17% expressed non-truncated ICN1 (NT-ICN1); and 20% had undetectable ICN1 (UD-ICN1), most lacked Notch1 mRNA. We confirmed the difference in Notch transcriptional activity and functional dependence between the UD-ICN1 and T-ICN1 subgroups and then compared their gene expression profiles to define pathways unique to the UD-ICN1 group. Gene set enrichment analyses revealed that UD-ICN1 T-LLs expressed higher levels of Klf9 and other transcription factors associated with a highly proliferative stage of normal T-cell development. siRNA knock-down studies demonstrated that Klf9 promoted proliferation of UD-ICN1 but not T-ICN1 T-LL cells. Collectively, these data demonstrate that Klf9 can regulate proliferation of Notch-independent T-LLs and suggest that Klf9 may provide a novel therapeutic target for human T-LLs lacking activating NOTCH1 mutations. Disclosures:No relevant conflicts of interest to declare.