CTL Activity Research Articles

Specific Gamma Delta T Cells for Cellular Immunotherapy of EBV-Associated Diseases after Allo-HSCT By T-Cell Receptor Gene Modification

Epstein-Barr virus (EBV) infection may result in a spectrum of diseases in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially post-transplant lymphoproliferative disease (PTLD). Furthermore, the clinical course of EBV-associated diseases often superimposes with graft-versus-host disease (GVHD) and that increases the difficulty of treatment. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTL) is recommended as one of the first-line therapy for PTLD. Engineering of T cell receptor (TCR)-transferred T lymphocytes could be an attractive strategy to obtain sufficient T cells with an antigen specificity of choice. But TCR-modified alpha beta T cells are restricted by major histocompatibility (MHC) class I expression and low affinity. Because gamma delta T cells (GD T cells) recognize target cells independent of human leukocyte antigen (HLA), the alpha beta TCR engineering of GD T cells forms a feasible strategy to generate antigen-specific effector T cells without HLA restriction. And regulatory GD T cells (GD Tregs) (one of the novel subset of GD T cells), which express Foxp3 and primarily belong to CD27+CD25high phenotype, might have application prospect in the treatment of GVHD. In order to investigate a novel strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, we obtained the monoclonal EBV-CTL clone HLA-A*0201/GLCTLVAML from the PROIMMUNE company (U.K.) and detected peptide-specific CTL activity in chromium release assays, then identified the EBV-CTL associated - TCR Valpha 15 and TCR Vbeta 1 chain genes, and subsequently cloned into the eukaryotic expression vector pIRES to construct the recombinant plasmid; We transferred the EBV specific recombinant plasmid to GD T cells and GD Tregs (generated in vitro by stimulating with anti-TCR gamma delta in the presence of TGF-beta and IL-2). The TCR gene-transduced groups had special TCR Vbeta 1 expression in fluorescence detection, quantitative PCR and western Blot analysis. TCR gene-transduced GD T cells had significant cytotoxicity against the EBV+ target cell lines (including toledo cells (HLA-A2 positive) (38.17±0.86%) and daudi cells (HLA-A2 negative) (61.74±1.84%)) compared with empty vector transfected GD T cells (toledo cells (30.15±0.89%); daudi cells (53.91±4.33%)) (P<0.001, P=0.006). TCR gene-transduced GD Tregs also had significant cytotoxicity against toledo cells (56.21±5.34%) and daudi cells (68.41±2.30%) compared with empty vector transfected GD Tregs (toledo cells (48.47±0.35%); daudi cells (56.23±2.04%)) (P=0.012, P<0.001). Both TCR gene-transduced GD T cells and GD Tregs had high levels of IFN-gamma in culture supernatants. Moreover, TCR gene-transduced GD Tregs had high levels of IL-10 in culture supernatants. Interestingly, TCR-modified GD Tregs showed significant suppressive effect on autologous CD4+ responder cells in CCK8 detection and CFSE assays. In conclusion, GD T cells or GD Tregs co-transduced with TCR alpha beta genes (from EBV CTL clone) could acquire antiviral capacity. Immunomodulatory function of GD Tregs could remain after TCR gene transfection. TCR modified GD Tregs might be an alternative and attractive strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, especially with GVHD. DisclosuresWu:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding. Li:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding.

The efficacy of tumor debulking surgery is improved by adjuvant immunotherapy using imiquimod and anti-CD40.

BackgroundTumor debulking surgery followed by adjuvant chemotherapy or radiotherapy is a standard treatment for many solid malignancies. Although this approach can be effective, it often has limited success against recurrent or metastatic cancers and new multimodality approaches are needed. Adjuvant immunotherapy is another potentially effective approach. We therefore tested the efficacy of the TLR7 agonist imiquimod (IMQ) combined with agonistic anti-CD40 in an incomplete debulking model of malignant mesothelioma.MethodsEstablished subcutaneous murine ABA-HA mesothelioma tumors in BALB/c mice were surgically debulked by 75% and treated with either: i) saline; ii) intratumoral IMQ; iii) systemic anti-CD40 antibody, or using a combination of IMQ and anti-CD40. Tumour growth and survival were monitored, and the role of anti-tumor CD4 and CD8 T cells in therapeutic responses was determined.ResultsThe combination therapy of partial debulking surgery, IMQ and anti-CD40 significantly delayed tumor growth in a CD8 T cell dependent manner, and promoted tumor regression in 25% of animals with establishment of immunological memory. This response was associated with an increase in ICOS+ CD8 T cells and tumor-specific CTL activity in tumor draining lymph nodes along with an increase in ICOS+ CD8 T cells in responding tumours.ConclusionsWe show that the post-surgical environment can be significantly altered by the co-administration of adjuvant IMQ and anti-CD40, resulting in strong, systemic anti-tumor activity. Both adjuvants are available for clinical use/trial, hence this treatment regimen has clear translational potential.

Interleukin 27 inhibits cytotoxic T-lymphocyte-mediated platelet destruction in primary immune thrombocytopenia

AbstractCytotoxic T-lymphocyte (CTL)-mediated platelet destruction and aberrant cytokine profiles play important roles in the pathogenesis of primary immune thrombocytopenia (ITP). Interleukin-27 (IL-27) has pleiotropic immunomodulatory effects. However, the effect of IL-27 on CTL activity in ITP has not been reported. In the present study, platelets from ITP patients were cultured with autologous CTLs in the presence of IL-27. We found that IL-27 could inhibit CTL-mediated platelet destruction. In these IL-27–treated CTLs, granzyme B and T-bet expression decreased significantly, whereas granzyme A, perforin, and eomesodermin were not affected. To further investigate the role of granzyme B in CTL-mediated platelet destruction, granzyme B inhibitor was added and platelet apoptosis was significantly inhibited. These results suggest that IL-27 negatively regulates CTL cytotoxicity toward platelets in ITP by decreasing granzyme B expression, which is associated with reduced T-bet expression. IL-27 may have a therapeutic role in treating ITP patients.

Anti-GITR agonist therapy intrinsically enhances CD8 T cell responses to chronic lymphocytic choriomeningitis virus (LCMV), thereby circumventing LCMV-induced downregulation of costimulatory GITR ligand on APC.

The costimulatory TNFR family member GITR can provide important survival signals for CD8 T cells. However, little is known about the regulation of this pathway during a chronic infection. In this study, we show that GITR ligand (GITRL) is maximally induced on APCs at day 2 post-lymphocytic choriomeningitis virus (LCMV) clone 13 infection, but is downregulated to below baseline levels by day 8 postinfection (p.i.), and remains so at the chronic stage of infection. At its peak, GITRL expression is highest on macrophages, with lower expression on conventional and plasmacytoid dendritic cells. GITR expression was highest on T regulatory cells but was also detected on Th1 and LCMV-specific CD8 T cells at day 8 p.i. and was maintained at low, but above baseline levels at the chronic stage of LCMV infection. As GITRL was limiting at the chronic stage of infection, we investigated the potential of therapeutic stimulation of GITR at this stage using agonistic anti-GITR Ab. Anti-GITR treatment at day 21 p.i. increased the frequency and number of LCMV-specific CD8 T cells, resulting in increased in vivo CTL activity and a concomitant decrease in viral load, despite the persistence of PD-1 expression. These effects of anti-GITR were CD8 T cell intrinsic, with no detectable effects on Th1 or T regulatory cells. In contrast to other TNFR agonists, such as anti-4-1BB, which can cause immune pathology, a single therapeutic dose of anti-GITR did not induce splenomegaly or increase serum alanine transaminase. These studies identify GITR as a promising therapeutic target for chronic infection.

Exogenous Addition of Arachidonic Acid to the Culture Media Enhances the Functionality of Dendritic Cells for Their Possible Use in Cancer Immunotherapy

The development of dendritic cell based vaccines is a promising approach in cancer immunotherapy. For their successful use in the clinics, the propagation and functionality of DCs is crucial. We earlier established a two-step method for the large scale generation of DCs from umbilical cord blood derived MNCs/CD34+ cells. This work aims at improving their functionality based on the following observations: in vitro generated DCs can be less efficient in migration and other functional activities due to lower eicosanoid levels. The production of eicosanoids from Arachidonic Acid (AA) can be hampered due to suppression of the enzyme phospholipase A2 by IL-4, an essential cytokine required for the differentiation of DCs. We hypothesized that exogenous addition of AA to the culture media during DC generation may result in DCs with improved functionality. DCs were generated with and without AA. The two DC sets were compared by phenotypic analysis, morphology and functional assays like antigen uptake, MLR, CTL assay and in vitro and in vivo migration. Though there were no differences between the two types of DCs in terms of morphology, phenotype and antigen uptake, AA+ DCs exhibited an enhanced in vitro and in vivo migration, T cell stimulatory capacity, CTL activity and significantly higher transcript levels of COX-2. AA+ DCs also show a favorable Th1 cytokine profile than AA- DCs. Thus addition of AA to the culture media is skewing the DCs towards the secretion of more IL-12 and less of IL-10 along with the restoration of eicosanoids levels in a COX-2 mediated pathway thereby enhancing the functionality of these cells to be used as a potent cellular vaccine. Taken together, these findings will be helpful in the better contriving of DC based vaccines for cancer immunotherapy.

Abstract A26: Monocyte depletion to amplify tumor vaccine immunity in a dog non-Hodgkin lymphoma model

Abstract Background. Spontaneously-developing B cell lymphoma in dogs closely resembles non-Hodgkin lymphoma (NHL) in humans in many respects, including tumor biology, predictors of tumor progression, and development of resistance to chemotherapy. The canine NHL model therefore provides a strong platform for evaluation of therapeutics for human NHL, including assessment of novel immunotherapeutic approaches. We reported recently that inflammatory monocytes exerted potent suppressive effects on vaccine responses and that monocyte depletion at the time of immunization could significantly enhance tumor vaccine immunity, as assessed by studies in rodent tumor models. The present study was conducted to determine whether the monocyte depletion approach could also effectively augment tumor vaccine immunity in the canine spontaneous NHL model. Approach. A pilot study involving 20 dogs with NHL was conducted to assess the vaccine enhancing effects of monocyte depletion. Autologous lymphoma vaccines were prepared from lymph node biopsy specimens obtained from dogs with multicentric B cell NHL. To prepare tumor vaccines, cell membrane antigens were extracted from the tumor tissues and mixed with a liposomal-TLR adjuvant, and the resulting autologous tumor vaccine was administered s.c. Dogs enrolled in the study were randomized to receive the lymphoma vaccine alone or the vaccine administered together with i.v. liposomal clodronate as a transient monocyte depleting agent. Tumor remission was first induced using doxorubicin and dogs each received a total of 6 tumor vaccines over an 18-week period, administered along with concurrent doxorubicin chemotherapy. Cellular and humoral immune responses were evaluated using PBMC. Results. Immunization with the autologous lymphoma vaccine induced measurable cellular immune responses, including increased IFN-g recall responses to tumor antigens, increased tumor-specific CTL activity, and increased anti-tumor antibody responses. Immunization combined with monocyte depletion induced significant enhancement of both cellular and humoral immune responses to vaccination, compared to dogs that received the tumor vaccine alone. However, augmentation of vaccine immunity waned at the completion of the vaccination period, and disease-free intervals were not significantly different between the vaccine only and vaccine + liposomal clodronate groups of dogs. Conclusions. We concluded that transient monocyte depletion at the time of vaccination significantly enhanced vaccine immunity in dogs with NHL, though the vaccine enhancing effects were transient. Additional studies of monocyte targeted therapeutics for tumor vaccine enhancement to generate more durable vaccine immunity appear to be warranted based on the results of this feasibility study in a canine model of NHL. Citation Format: Steven Dow, Leah Mitchell, Amanda Guth, Dan Regan, Jenna Burton, Robyn Elmslie. Monocyte depletion to amplify tumor vaccine immunity in a dog non-Hodgkin lymphoma model. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A26.

A loss of antitumor therapeutic activity of CEA DNA vaccines is associated with the lack of tumor cells' antigen presentation to Ag-specific CTLs in a colon cancer model

Human colon cancers express carcinoembryonic antigen (CEA). Thus, CEA has been considered as a potential vaccine target for immune therapy against colon cancer. In this study, CEA DNA vaccines plus anti-4-1BB Abs treatment was found to increase Ag-specific CTL activity and antitumor protective responses to MC32 cells. However, CEA DNA vaccines alone displayed few antitumor therapeutic effects while significantly inducing Ag-specific CTL responses. Anti-4-1BB Abs alone displayed antitumor therapeutic effects. Intratumoral electroporation with IL-12 cDNA also showed antitumor therapeutic activity against MC32 cells in a CD8+ T cell-dependent and CEA-non-specific manner, suggesting that established MC32 cells are still susceptible to CTL-mediated killing. Finally, our in vitro assays (Western blot assay, IFN-γ, CTL and apoptosis assays, FACS analysis) and animal studies demonstrated that a lack of antitumor therapeutic activity of CEA DNA vaccines might result from acquisition of tumor cell resistance to Ag-specific CTL-mediated killing through the loss of tumor cells' antigen presentation to Ag-specific CTLs. Taken together, these data show that MC32 cells may resist CEA DNA vaccination by their loss of antigen presentation to CEA-specific CTLs in the therapeutic model.

Abstract 636: Sub-lethal irradiation of diverse human carcinoma cells imparts enhanced and sustained expression of important modulators of effector CTL activity

Abstract Current cancer immunotherapy (CIT) approaches can efficiently introduce anti-tumor effector T-cells in patients, however, tumor cells can be immunosuppressive and directly inhibitory to T-cell activity. Previous data suggests that human tumor cells surviving radiation are phenotypically modulated making them better targets for CTLs. We examined several tumor cell lines (colorectal, breast and prostate) for their response to various doses of radiation (0-10Gy). Experiments quantified changes in the expression of genes that could result in altered effector CTL activity (OX40L, 41BBL, PD-L1, ICOSL and CD70) against tumor cells. Following irradiation, changes in expression of effector costimulatory molecules was examined in surviving and proliferating tumor cells. In human tumor cells expressing similar levels of major histocompatibility complex class I (MHC-I) and tumor associated antigens (TAA) following radiation, differences in CTL activity correlated with modulation of two positive costimulators, OX40L and 41BBL. The genes upregulated in our study (41BBL and OX40L) provide positive signals to killer T-cells that can attack tumor cells. Increased expression occurred when radiation was delivered either as a single dose or in fractions and could be observed as long as 7-days post-irradiation. We also found that radiation could alter gene expression in tumor cells via epigenetic mechanisms such as histone modifications of specific promoters. Furthermore we discovered that altered expression of OX40L and 41BBL (but not CD70, ICOSL or PD-L1) correlated with enhanced killing of irradiated tumor cells by both CEA and MUC-specific CTLs in cytotoxicity assays and this lysis was reversed by costimulatory blocking or gene-knockdown. We also observed enhanced activation and survival of CTLs exposed to irradiated tumor cell lines as well as exposed to ex vivo irradiated CD326+ tumor cells isolated from patient tumors. Overall, the results of our studies suggest that radiation can be used to make human tumors more amenable to effector CTL attack. Significance: The full role of IR as an independent immune-enhancer, in the absence of cell death or in radio-resistant tumors, remains unclear. Our data addresses a critical barrier to progress in improving combination radiation-cancer immunotherapy (RT-CIT) strategies by identifying mechanisms responsible for increased killing of irradiated tumor cells by effector CTLs. This “immunogenic modulation” of tumor cells is a mechanism different from ICD (requiring death of tumor cells and subsequent antigen processing and presentation by dendritic cells) that could be invoked independently, or to complement ICD approaches. This approach represents an alternate way of triggering important T-cell signal pathways that does not require the use of agonist antibodies. Citation Format: Anita Kumari, Orpha Rachel Mott, Ercan Cacan, Susanna F. Greer, Charlie T. Garnett. Sub-lethal irradiation of diverse human carcinoma cells imparts enhanced and sustained expression of important modulators of effector CTL activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 636. doi:10.1158/1538-7445.AM2014-636

Abstract 669: Development of MGD007, a gpA33 x CD3 bi-specific DART for T-cell immunotherapy of metastatic colorectal cancer

Abstract Introduction: Encouraging clinical responses have been observed through various strategies designed to harness T-cells for their anti-tumor properties, including immune checkpoint inhibition, chimeric antigen receptor-expressing T-cells or bispecific molecules designed to co-engage T-cells and cancer cells. Furthermore, a positive correlation has been observed in colorectal cancer between the degree and type of T-cell infiltration and prognosis suggesting that T-cell recruitment strategies may be particularly advantageous in this type of cancer. We have developed MGD007, a Dual-Affinity Re-Targeting (DART®) protein designed to redirect T-cells to target gpA33 expressing colon cancer. MGD007 has enhanced pharmacokinetic properties via incorporation of a neonatal FcR-binding Fc domain. The gpA33 target was selected based on its ubiquitous expression in colorectal cancer including reactivity with putative cancer stem cell populations (Li 2013, AACR #3763). To enable preclinical toxicokinetics and dose optimization, MGD007 was designed to cross-react with non-human primates. Methods: MGD007 was stably expressed in CHO cells and purified to homogeneity via a standard antibody-purification platform; in vitro functional studies were performed with a range of colorectal cancer cell lines and primary human T-cells; tumor growth inhibition studies were performed in NOD-SCID mice co-implanted with Colo205 and human T-cells (1:1 E:T ratio) and treated IV with MGD007; pharmacokinetic analyses were performed in cynomolgus monkeys. Results: MGD007 displays the anticipated bispecific binding properties and mediates potent lysis of gpA33-positive - but not gpA33-negative cancer cell lines through recruitment of either human or cynomolgus monkey T-cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Both CD8 and CD4 T-cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme levels. Xenograft studies showed tumor growth inhibition at doses as low as 4ug/kg. In cynomologus monkeys, 4 weekly doses of 200ug/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Conclusions: MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design. Furthermore it displays favorable PK in cynomolgus monkeys supporting convenient dosing. Taken together, these data support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Citation Format: Paul A. Moore, Ralph Alderson, Kalpana Shah, Yinhua Yang, Steve Burke, Hua Li, Valentine Ciccarone, Ezio Bonvini, Syd Johnson. Development of MGD007, a gpA33 x CD3 bi-specific DART for T-cell immunotherapy of metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2014-669

Abstract LB-150: Local maturation and inhibition of antitumor CD8 T-cell responses in a model of immunoevasive breast cancer

Abstract The purpose of this study is to investigate the cell interactions and mechanisms that limit anti-tumor CD8 T cell responses in tumor microenvironments. We used cutting-edge multiphoton imaging of immunoevasive tumors to determine the behavior and anti-tumor responses of CD8 T cells. This was achieved by adoptively transferring T cells into a modified MMTV-PyMT breast tumor model (mouse mammary tumor virus promoter-driven, polyoma middle T antigen-induced luminal breast cancer). In this model, tumor cells expresses ovalbumin as tumor-antigen and mCherry as a fluorescent markers, thus allowing immunologic assessment and intravital imaging of T cell activity against tumor cells in and ex vivo. Furthermore, we were able to investigate the effect of antigen-presenting immune cells and immunomodulatory treatment regimens on anti-tumor T cell activity in and ex vivo. In this work we found that newly arriving tumor-specific T cells undergo defined phases of maturation within the tumor microenvironment. First, after arriving to the tumor site, T cells engage in close interactions with antigen-presenting cells and exhibit reduced motility and rounded morphology. After this initial phase, T cells dramatically increase motility and survey the tumor microenvironment, however they do not form stable interactions with other cell types and further do not engage tumor cells. In contrary to other publications, swarming T cells do not express surface markers associated with exhaustion or anergy, and exhibit extensive in situ proliferation and ex vivo CTL activity. The capability of tumor CD8 T cells to kill targets ex vivo conflicts with the absence of tumor cell death in vivo. Additionally, individual immune cell populations isolated from tumors or tumor culture supernatants do not exhibit T cell suppressive capability when added as supplements to killing assays. Our results define sequential fates for T cells entering tumor microenvironments, suggesting that tumor-infiltrating lymphocytes exist in distinct ‘states’ depending on the extent to which tumor antigen-presenting cells have matured them. Further, T cell suppression appears to be a local phenomenon and cannot be achieved by soluble factors or a specific cell type alone. This may suggest that combinations of therapies that individually target the different maturation states are required to achieve therapeutic effects. Citation Format: Bijan A. Boldajipour, Matthew F. Krummel. Local maturation and inhibition of antitumor CD8 T-cell responses in a model of immunoevasive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-150. doi:10.1158/1538-7445.AM2014-LB-150

Inhibition of mitogen-activated protein kinase pathway can induce upregulation of human leukocyte antigen class I without PD-L1-upregulation in contrast to interferon-γ treatment.

Recently, we reported that human leukocyte antigen (HLA) class I expression is predominantly regulated by the mitogen-activated protein kinase (MAPK) pathway as one of the oncogenic regulations of HLA class I expression. In the present study, we examined mechanisms of how HLA class I and PD-L1 are regulated by MAPK inhibitors and interferon-γ (IFN-γ). Furthermore, we evaluated the expression of major signal transduction molecules by Western blot and anti-tumor CTL activity by a cytotoxic assay when HLA class I and PD-L1 were modulated by MAPK inhibitors and/or IFN-γ. As a result, we confirmed, as a more general phenomenon, that the inhibition of MAPK could upregulate HLA class I expression in a panel of human solid tumors (n = 26). Of note, we showed that MAPK inhibitors act on the upregulation of HLA class I expression through a different pathway from IFN-γ; there was an additive effect in the upregulation of HLA class I when treated with the combination of MAPK inhibitors and IFN-γ, and there was no overlapping activation of JAK2/STAT1 and Erk1/2 molecules when treated with either IFN-γ or MAPK inhibitors. Furthermore, we showed that IFN-γ–treatment impaired the tumor-specific CTL activity due to the upregulation of PD-L1 in spite of the upregulation of HLA class I, while MAPK inhibitors can augment the tumor-specific CTL activity due to the upregulated HLA class I without PD-L1 alterations. In conclusion, in addition to the original anti-proliferative activity, MAPK inhibitors may work toward the enhancement of T-cell-mediated anti-tumor immunity through the upregulation of HLA class I without the upregulation of PD-L1.

CD40 ligand preferentially modulates immune response and enhances protection against influenza virus.

CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L(-/-) and CD4(-/-)) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8(+) T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L(-/-) and CD4(-/-) mice revealed that the protection was indeed CD40L mediated but CD4(+) T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.

PTPN2 restrains CD8+ T cell responses after antigen cross-presentation for the maintenance of peripheral tolerance in mice

Antigen cross-presentation by dendritic cells is crucial for priming cytotoxic CD8+ T cells to invading pathogens and tumour antigens, as well as mediating peripheral tolerance to self-antigens. The protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor (TCR) signalling and tunes CD8+ T cell responses in vivo. In this study we have examined the role of PTPN2 in the maintenance of peripheral tolerance after the cross-presentation of pancreatic β-cell antigens. The transfer of OVA-specific OT-I CD8+ T cells (C57BL/6) into RIP-mOVA recipients expressing OVA in pancreatic β-cells only results in islet destruction when OVA-specific CD4+ T cells are co-transferred. Herein we report that PTPN2-deficient OT-I CD8+ T cells transferred into RIP-mOVA recipients acquire CTL activity and result in β cell destruction and the development of diabetes in the absence of CD4+ help. These studies identify PTPN2 as a critical mediator of peripheral T cell tolerance limiting CD8+ T cell responses after the cross-presentation of self-antigens. Our findings reveal a mechanism by which PTPN2 SNPs might convert a tolerogenic CD8+ T cell response into one capable of causing the destruction of pancreatic β-cells. Moreover, our results provide insight into potential approaches for enhancing T cell-mediated immunity and/or T cell adoptive tumour immunotherapy.

Fucoidan can function as an adjuvant in vivo to enhance dendritic cell maturation and function and promote antigen-specific T cell immune responses.

Fucoidan, a sulfated polysaccharide purified from brown algae, has a variety of immune-modulation effects, including promoting antigen uptake and enhancing anti-viral and anti-tumor effects. However, the effect of fucoidan in vivo, especially its adjuvant effect on in vivo anti-tumor immune responses, was not fully investigated. In this study, we investigated the effect of fucoidan on the function of spleen dendritic cells (DCs) and its adjuvant effect in vivo. Systemic administration of fucoidan induced up-regulation of CD40, CD80 and CD86 expression and production of IL-6, IL-12 and TNF-α in spleen cDCs. Fucoidan also promoted the generation of IFN-γ-producing Th1 and Tc1 cells in an IL-12-dependent manner. When used as an adjuvant in vivo with ovalbumin (OVA) antigen, fucoidan promoted OVA-specific antibody production and primed IFN-γ production in OVA-specific T cells. Moreover, fucoidan enhanced OVA-induced up-regulation of MHC class I and II on spleen cDCs and strongly prompted the proliferation of OVA-specific CD4 and CD8 T cells. Finally, OVA immunization with fucoidan as adjuvant protected mice from the challenge with B16-OVA tumor cells. Taken together, these results suggest that fucoidan can function as an adjuvant to induce Th1 immune response and CTL activation, which may be useful in tumor vaccine development.

USP18 is crucial for IFN-γ-mediated inhibition of B16 melanoma tumorigenesis and antitumor immunity.

BackgroundInterferon (IFN)-γ-mediated immune response plays an important role in tumor immunosurveillance. However, the regulation of IFN-γ-mediated tumorigenesis and immune response remains elusive. USP18, an interferon stimulating response element, regulates IFN-α-mediated signaling in anti-viral immune response, but its role in IFN-γ-mediated tumorigenesis and anti-tumor immune response is unknown.MethodIn this study, USP18 in tumorigenesis and anti-tumor immune response was comprehensively appraised in vivo by overexpression or downregulation its expression in murine B16 melanoma tumor model in immunocompetent and immunodeficient mice.ResultsEctopic expression or downregulation of USP18 in B16 melanoma tumor cells inhibited or promoted tumorigenesis, respectively, in immunocompetent mice. USP18 expression in B16 melanoma tumor cells regulated IFN-γ-mediated immunoediting, including upregulating MHC class-I expression, reducing tumor cell-mediated inhibition of T cell proliferation and activation, and suppressing PD-1 expression in CD4+ and CD8+ T cells in tumor-bearing mice. USP18 expression in B16 melanoma tumor cells also enhanced CTL activity during adoptive immunotherapy by prolonging the persistence and enhancing the activity of adoptively transferred CTLs and by reducing CTL exhaustion in the tumor microenvironment. Mechanistic studies demonstrated that USP18 suppressed tumor cell-mediated immune inhibition by activating T cells, inhibiting T-cell exhaustion, and reducing dendritic cell tolerance, thus sensitizing tumor cells to immunosurveillance and immunotherapy.ConclusionThese findings suggest that stimulating USP18 is a feasible approach to induce B16 melanoma specific immune response.

Mycobacterium tuberculosis multidrug resistant strain M induces an altered activation of cytotoxic CD8+ T cells.

In human tuberculosis (TB), CD8+ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8+ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4+ and CD8+ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8+ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4+ and CD8+ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8+ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8+ T cells as well as CD4+ T cells collaboration.

The role of CD99 in T cell activation, signaling, and immunological synapse formation (IRM11P.760)

Abstract Cytotoxic T lymphocytes (CTLs) provides a defense against virus-infected and tumorigenic cells. The recognition of antigen-presenting cells (APCs) for T cells and the association with them are essential in the formation of the immunological synapse (IS) which triggers T cell activation. Although various proteins that stabilize the IS structure for delivering appropriate signals in T cells have been identified, the role of adhesion molecule CD99 in CTL activation and IS formation has not been investigated. Here we found that CD99 in CTLs participates in T cell activation, proliferation, and effector differentiation by inducing of cluster formation between T cells and APCs. Interestingly, it is shown that CD99 is co-localized more at the peripheral supramolecular activating cluster (pSMAC) region than the central supramolecular activating cluster (cSMAC) in the IS, indicating the involvement of CD99 in IS formation. The absence of CD99 in CD8+ T cells resulted in slower formation of T-APC conjugates than WT CTLs. In addition, Akt-mediated signaling, followed by activation, proliferation, and cytokine production, was delayed in CD99KO CTLs upon TCR stimulation. Results presented here demonstrate for the first time that CD99 is recruited to the IS during APC and T cell interaction and plays an important role in subsequent T cell activation.

NKT cells act as helper T cells to cytotoxic T cells by direct recognition of CD1d on T cells (IRC8P.487)

Abstract Cytotoxic T lymphocytes (CTLs), known as CD8+ T cells, recognize their targets by binding to antigens associated with MHC class I. NKT cells recognize foreign glycolipid antigens and endogenous glycolipid antigens presented by CD1d which is an MHC class I- like molecule. NKT cells have been shown to be essential for the optimal activation of CTLs. In this study, we investigated the mechanism how NKT cells provide help for the CTL responses. We observed significantly higher production of IFN-γ by antigen-stimulated OT-1 CD8+ T cells in the presence of NKT cells. This enhanced CTL activation by NKT cells was observed even in the antigen presenting cell (APC)-free conditions. We also found that CD8+ T cells start to up-regulate surface expression of CD1d reaching highest level at around 18 hours upon antigen recognition. These results suggest that not only APC, but also activated CTLs receive direct help from NKT cells by increasing CD1d expression on their cell surface when they recognize cognate antigens.

An aqueous extract of Limoniastrum guyonianum gall induces anti-tumor effects in melanoma-injected mice via modulation of the immune response

The objectives of this study were to evaluate the in vitro and in vivo anti-tumor potential of the aqueous gall extract (G extract) from Limoniastrum guyonianum and to elucidate its immunological mechanisms, in part, by assessing its effects on the growth of transplanted tumors and the immune response in these tumor-bearing mice. Here, mice were inoculated with B16F10 mouse tumor cells and then treated intraperitoneally with G extract at 25 or 50mg extract/kgBW for 7, 14, or 21days. At each timepoint, effects of the extract on the tumor growth, splenocytes proliferation, NK cell activity, and CTL activity among splenocytes isolated from the mice were measured. G extract-induced tumor growth inhibition was associated with characteristic apoptotic changes in the tumor cells, like nuclear condensation. In addition, the extract inhibited melanin synthesis and tyrosinase activity among melanoma cells in a concentration-related manner. G extract did not only significantly inhibit the growth of the transplantable tumor, but also remarkably increased splenocytes proliferation and both NK and CTL activities in tumor-bearing mice. The extract was also seen to have promoted lysosomal activity of host macrophages and gave rise to enhanced cellular anti-oxidant activity in several cell types in mice.

New C4- and C1-derivatives of furo[3,4-c]pyridine-3-ones and related compounds: Evidence for site-specific inhibition of the constitutive proteasome and its immunoisoform

A set of 18 new C4 and C1 derivatives of nor-cerpegin (1,1-dimethyl furo[3,4-c]pyridine-3-one), 6 model compounds (γ- and δ-lactones) and 20 furo- or thieno[2,3-d]-pyrimidine-4-one related compounds were designed and synthesized. Each compound was assayed for inhibition of CT-L, T-L and PA proteolytic activities of 20S constitutive proteasome (c20S). Most performant compounds were also assayed on 20S immunoproteasome (i20S). Compound 10 with a benzylamino group at C4 and dimethylated at C1 of the furopyridine ring was the most efficient PA site-specific inhibitor of the c20S (IC50cPA of 600nM) without noticeable inhibition of the i20S PA site (iPA). In silico docking assays for 10 at the iPA catalytic site revealed the absence of poses normally observed for this compound and related ones at the constitutive PA site (cPA). The thieno[2,3-d]pyrimidine-4-one 40 was T-L site-specific with a mild inhibition of both c20S and i20S in vitro (IC50cT-L of 9.9μM and IC50iT-L of 6.7μM). In silico docking assays of 40 at T-L sites of c20S and i20S revealed almost identical first rank poses in the two types of sites with no possibility left for nucleophilic attack by Thr1 as observed for the fused furopyridine-3-one 10.