T-cell Induction Research Articles

Helicobacter pylori and Extragastric Diseases.

The Gram-negative bacterium Helicobacter pylori is predominantly known for its tight association with peptic ulcer disease and gastric cancer development. However, recent epidemiological and experimental evidence suggests that chronic infection with H. pylori may at the same time be beneficial to the host by conferring protection against gastroesophageal diseases, asthma, other allergic disease manifestations and inflammatory bowel diseases (IBD). In this chapter, we summarize the epidemiological data that are available to date to support or refute a possible inverse correlation of H. pylori infection with various extragastric diseases. We further examine and discuss the experimental evidence, generated mostly in mouse models of allergic diseases and IBD, showing that these disorders fail to develop in the presence of H. pylori. The proposed mechanisms of the protective effects of H. pylori, which appear to involve the induction of regulatory T-cells (Tregs) with highly suppressive activity, are presented and explained.

FRI-296 - Myeloid-Derived Suppressor Cells: key players mediating immunosuppression via induction of Regulatory T-cell by TGFβ and IL-10 driven pathways in chronic HBV infection

FRI-296 - Myeloid-Derived Suppressor Cells: key players mediating immunosuppression via induction of Regulatory T-cell by TGFβ and IL-10 driven pathways in chronic HBV infection

In Vitro Induction of Human Regulatory T-Cells (iTregs) Using Conditions of Low Tryptophan Plus Kynurenines

Thymic regulatory T cells (tTreg) or in vitro induced Tregs (iTregs) adoptive transfer suppresses murine acute graft-versus-host disease (GVHD). Previously we demonstrated that plasmacytoid dendritic cell (pDC) indoleamine 2,3-dioxygenase (IDO) fosters the development of human iTregs through tryptophan (Trp) depletion and production of catabolites including kynurenines (Kyn). IDO is an immunosuppressive enzyme implicated in maternal-fetal acceptance, tumor immunity, and autoimmunity. IDO is highly up-regulated in the colon of mice and patients with GVHD; in rodents, the lack of IDO leads to increased colon pathology and accelerated lethality. Because pDCs are rare and difficult to purify and expand, we sought to determine whether an optimally suppressive iTreg could be generated using clinically amenable conditions that mimicked pDCs support (i.e. Low Trp+Kyn).Naïve human CD4+ T cells purified with cGMP-compatible reagents and expanded in vitro using an anti-CD3-loaded artificial APC expressing CD86 under conditions of low Trp + kynurenines suppress in vitro T cell proliferation equivalently to tTreg (A) (Fig 1). The calculated yield of Low Trp+Kyn iTreg (~1x1010) produced from an apheresis product is ~10-fold greater than similarly expanded cord blood-derived tTreg that decreased GVHD severity in the clinic (B). Low Trp+Kyn iTreg have a phenotype distinct from tTreg, including: higher CD25 expression, lower Foxp3 expression, and no Helios expression (consistent with the non-thymus origin of iTregs) (C).Since Trp depletion was important for iTreg generation, we determined whether iTregs were under metabolic stress. Surprisingly, bioenergetics profiling demonstrated that Low Trp+Kyn iTreg maintain a remarkably high metabolic rate, with both oxidative phosphorylation and glycolytic pathways higher than that observed in expanded Teffectors (Teff) and tTregs (D). High metabolic rates had functional consequences, as Low Trp+Kyn iTreg were exquisitely dependent on IL-2, which drives the expression of genes needed for glycolytic metabolism. In addition, high basal oxidative phosphorylation rates correlated with increased mitochondrial membrane potential and ROS production (not shown) and decreased viability.A xenogeneic model of GVHD was used to assess the in vivo efficacy of Low Trp+Kyn iTreg. Because Low Trp+Kyn iTreg were so sensitive to IL-2 withdrawal in vitro, mice were also treated ± low dose IL-2 for 9 days (IP, 25kU/day). While low dose IL-2 prolonged in vivo Low Trp+Kyn iTreg persistence and improved survival compared to no IL-2, survival was not significantly increased compared to GVHD mice receiving only PBMC (E). To determine whether increasing the duration of IL-2 therapy would further increase Low Trp+Kyn iTreg persistence and efficacy, the experiment was repeated (with the addition of a cohort of mice receiving PBMC + IL-2 only), and IL-2 treatment was continued indefinitely (F). However, prolonged IL-2 therapy enhanced Teff expansion, and all animals receiving exogenous IL-2 (either PBMC only or with Low Trp+Kyn iTreg) were deceased by day 18. Studies are in progress to determine whether lower doses of IL2 (i.e. 2.5 or 10k U/day) can preferentially foster iTreg expansion and efficacy in suppressing xenogeneic GVHD over Teff expansion and exacerbation of disease. [Display omitted] DisclosuresHippen:University of Minnesota: Other: holds patents for the production and use of iTregs for clinical trials. Riley:Tmunity Therapeutics: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; University of Pennsylvania: Patents & Royalties: hold patents for the production and use of iTregs for clinical trials. June:Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties. Kean:Juno Therapeutics, Inc: Research Funding. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Identification of genetic pathways driving Ebola virus disease in humans and targets for therapeutic intervention

Introduction: LCKgene, also known as lymphocyte-specific proto-oncogene, is expressed in lymphocytes, and associated with coordinated expression of MHC class I and II in response to physiological stimuli, mediated through a combined interaction of promoters, suppressors, and enhancers. Differential usage ofLCKpromoters, transcribes dysfunctional transcript variants leading to leukemogenesis and non-induction of MHC class I gene variants. Viruses use C-type lectins, likeCD209,to penetrate the cell, and inhibit Pattern Recognition Receptors (PRR), hence evading immune destruction. Given that Ebolavirus (EBOV) disease burden could result from a dysfunctional LCK pathway, identification of the genetic pathway leading to proper immune induction is a major priority.Methods:Data for EBOV related virus samples were obtained from Gene Expression Omnibus database and RMEAN information per gene per sample were entered into a table of values. R software v.3.3.1 was used to process differential expression patterns across samples forLCK, CD209and immune-related genes. Principal component analysis (PCA) using ggbiplot v.0.55 was used to explain the variance across samples.Results:Data analyses identified three viral clusters based on transmission patterns as follows:LCK-CD209dependent, LCK-dependent specific to EBOV, and CD209 dependent. Compared to HLA class II gene variants, HLA class I (A, B and C) variants were <2 fold expressed, especially for EBOV samples. PCA analyses classifiedTYRO3,TBK1andLCKgenes independent of the data, leading to identification of a possible pathway involvingLCK, IL2, PI3k, TBK1, TYRO3andMYBgenes with downstream induction of immune T-cells.Discussion: This is the first study undertaken to understand the non-functional immune pathway, leading to EBOV disease pathogenesis and high fatality rates. Our lab currently exploits, through cutting edge genetic technology to understand the interplay of identified genes required for proper immune induction. This will guide antiviral therapy and possible markers for viral disease identification during outbreaks.

High-Parameter Mass Cytometry (CyTOF) Evaluation of Relapsed/Refractory Multiple Myeloma (MM) Pts (Pts) Treated with Daratumumab Supports Immune Modulation As a Novel Mechanism of Action

High-Parameter Mass Cytometry (CyTOF) Evaluation of Relapsed/Refractory Multiple Myeloma (MM) Pts (Pts) Treated with Daratumumab Supports Immune Modulation As a Novel Mechanism of Action

The "ins and outs" of complement-driven immune responses.

The complement system represents an evolutionary old and critical component of innate immunity where it forms the first line of defense against invading pathogens. Originally described as a heat-labile fraction of the serum responsible for the opsonization and subsequent lytic killing of bacteria, work over the last century firmly established complement as a key mediator of the general inflammatory response but also as an acknowledged vital bridge between innate and adaptive immunity. However, recent studies particularly spanning the last decade have provided new insights into the novel modes and locations of complement activation and highlighted unexpected additional biological functions for this ancient system, for example, in regulating basic processes of the cell. In this review, we will cover the current knowledge about complement's established and novel roles in innate and adaptive immunity with a focus on the functional differences between serum circulating and intracellularly active complement and will describe and discuss the newly discovered cross-talks of complement with other cell effector systems particularly during T-cell induction and contraction.

AHR Activation Is Protective against Colitis Driven by T Cells in Humanized Mice

Existing therapies for inflammatory bowel disease that are based on broad suppression of inflammation result in variable clinical benefit and unwanted side effects. A potential therapeutic approach for promoting immune tolerance is the invivo induction of regulatory Tcells (Tregs). Here we report that activation of the aryl hydrocarbon receptor using the non-toxicagonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces human Tregs invitro that suppress effector Tcells through a mechanism mediated by CD39 and Granzyme B. We then developed a humanized murine system whereby human CD4+ Tcells drive colitis upon exposure to 2,4,6-trinitrobenzenesulfonic acid and assessed ITE as a potential therapeutic. ITE administration ameliorated colitis in humanized mice with increased CD39, Granzyme B, and IL10-secreting human Tregs. These results develop an experimental model to investigate human CD4+ T responses invivo and identify the non-toxic AHR agonist ITE as a potential therapy for promoting immune tolerance in the intestine.

Infant milk formulas differ regarding their allergenic activity and induction of T-cell and cytokine responses.

BackgroundSeveral hydrolyzed cow's milk (CM) formulas are available for avoidance of allergic reactions in CM‐allergic children and for prevention of allergy development in high‐risk infants. Our aim was to compare CM formulas regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T‐cell proliferation, and cytokine secretion.MethodsA blinded analysis of eight CM formulas, one nonhydrolyzed, two partially hydrolyzed (PH), four extensively hydrolyzed (EH), and one amino acid formula, using biochemical techniques and specific antibody probes was conducted. IgE reactivity and allergenic activity of the formulas were tested with sera from CM‐allergic patients (n = 26) in RAST‐based assays and with rat basophils transfected with the human FcεRI, respectively. The induction of T‐cell proliferation and the secretion of cytokines in Peripheral blood mononuclear cell (PBMC) culture from CM allergic patients and nonallergic individuals were assessed.ResultsImmune‐reactive α‐lactalbumin and β‐lactoglobulin were found in the two PH formulas and casein components in one of the EH formulas. One PH formula and the EH formula containing casein components showed remaining IgE reactivity, whereas the other hydrolyzed formulas lacked IgE reactivity. Only two EH formulas and the amino acid formula did not induce T‐cell proliferation and proinflammatory cytokine release. The remaining formulas varied regarding the induction of Th2, Th1, and proinflammatory cytokines.ConclusionOur results show that certain CM formulas without allergenic and low proinflammatory properties can be identified and they may also explain different outcomes obtained in clinical studies using CM formulas.

In VivoFluorescence Imaging of the Activity of CEA TCB, a Novel T-Cell Bispecific Antibody, Reveals Highly Specific Tumor Targeting and Fast Induction of T-Cell–Mediated Tumor Killing

CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ε upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts. CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB. Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell-dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells. Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB-mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417-27. ©2016 AACRSee related commentary by Teijeira et al., p. 4277.

The engagement of oral-associated lymphoid tissues during oral versus gastric antigen administration

The role of oral-associated lymphoid tissues during induction of oral tolerance still remains elusive. Therefore, the aim was to compare T-cell activation and induction of tolerance to ovalbumin (OVA) presented through either of two routes; deposited into the oral cavity, or the stomach, thereby bypassing the oral cavity. OVA was administered by the oral or gastric route to BALB/c mice that had received OVA-specific DO11.10+ CD4(+) T cells, stained with CellTrace(™) Violet dye, through intravenous injection. Proliferating OVA-specific T cells were detected in the nose-associated lymphoid tissues (NALT) and the cervical, mesenteric and peripheral lymph nodes at different time-points following OVA exposure. OVA-specific T-cell proliferation was initially observed in the NALT 1hr after oral, but not gastric, administration. However, at day 1, proliferation at this site was also detected after gastric administration and profound proliferation was observed at all sites by day 4. For the oral route the degree of proliferation observed was lower in the peripheral lymph nodes by day 4 compared with the other sites. These results demonstrate a similar activation pattern achieved by the two routes. However, the NALT distinguishes itself as a site of rapid T-cell activation towards fed antigens irrespective of feeding regimen. To evaluate induction of tolerance a semi-effective OVA dose was used, to detect differences in the degree of tolerance achieved. This was performed in a model of OVA-induced airway hypersensitivity. No differences in tolerance induction were observed between the two administration routes.

Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection.

An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T-cells were only induced by vaccination when there was a sequence mismatch between the autologous virus and the vaccine immunogen. However, these T-cells were not cross-reactive with the endogenous viral variant epitopes. Conversely, when there was complete homology between the immunogen and circulating virus at a given epitope T-cells were not induced. T-cell induction following vaccination had no significant impact on HCV viral load. In vitro T-cell culture experiments identified the presence of T-cells at baseline that could be expanded by vaccination; thus, HCV-specific T-cells may have been expanded from pre-existing low-level memory T-cell populations that had been exposed to HCV antigens during natural infection, explaining the partial T-cell dysfunction. In conclusion, vaccination with ChAd3-NSmut and MVA-NSmut prime/boost, a potent vaccine regimen previously optimized in healthy volunteers was unable to reconstitute HCV-specific T-cell immunity in HCV infected patients. This highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure.

Abstract 2346: Intratumoral administration of OncoVEXmGM-CSF results in local innate immune alterations and induces systemic anti-tumor effects in syngeneic mouse tumor models

Abstract Talimogene laherparepvec, is a modified oncolytic herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer cells. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors, with lytic cell destruction promoting the local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally and is designed to recruit and stimulate antigen presenting cells to further enhance systemic antitumor immune response. While available evidence suggests that talimogene laherparepvec can induce effects in distant tumors via an immune-mediated effect, additional mechanistic evidence is being sought to help better understand the putative systemic effect. For preclinical mechanism of action studies we employed OncoVEXmGM-CSF, an HSV-1 virus modified in the same manner as talimogene laherparepvec except that murine GM-CSF is expressed. We utilized this virus to further dissect the underlying innate and adaptive immunity following viral administration in syngeneic tumor models. Intratumoral administration of OncoVEXmGM-CSF into established murine tumors induced regressions in the injected lesion but also resulted in significant anti-tumor effects in contralateral (uninjected) lesions. We have previously demonstrated by immunohistochemistry that the systemic effects observed in the contralateral lesions are not driven by systemic spread of the virus, but instead correlate with the infiltration of CD3+ T cell populations. Here we report the full phenotypic characterization of immune infiltrates by flow cytometry in both the injected and contralateral lesions. In a time-course fashion, this characterization revealed the initial infiltration of the injected tumor by innate effector cells and a concomitant induction of a potent type I interferon response. This in turn drives the local release of proinflammatory cytokines and chemokines, resulting in the recruitment of adaptive immune subsets to the injected tumor and enhancing systemic anti-tumor reactivity. Additionally, both the primary oncolytic replication of the virus and the activation of type I interferon pathway are directly correlated with STING activity. In conclusion, in vitro and in vivo observations suggest that intratumoral injection of OncoVEXmGM-CSF activates multiple immune-mediated mechanisms of action leading to T-cell activation and induction of anti-tumor immunity in mice. Citation Format: Keegan Cooke, Karen Fitzgerald, Becky Yang, Juan Estrada, Brian Belmontes, Pedro Beltran, Achim K. Moesta. Intratumoral administration of OncoVEXmGM-CSF results in local innate immune alterations and induces systemic anti-tumor effects in syngeneic mouse tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2346.

Exploiting in situ antigen generation and immune modulation to enhance chemotherapy response in advanced melanoma: A combination nanomedicine approach

Therapeutic anticancer vaccine development must address a number of barriers to achieve successful tumor specific killing, including effective antigen presentation and antigen-specific T-cell activation to mediate cytotoxic cellular effects, inhibition of an immune-suppressive tumor microenvironment in order to facilitate and enhance CTL activity, and induction of memory T-cells to prolong tumor rejection. While traditional as well as modern vaccines rely upon delivery of both antigen and adjuvant, a variety of clinically relevant cancers lack ideal immunogenic antigens. Building upon recent efforts, we instead chose to exploit chemotherapy-induced apoptosis to allow for in situ antigen generation in a combination, nanomedicine-based approach. Specifically, lipid-coated cisplatin nanoparticles (LPC) and CpG-encapsulated liposomes (CpG-Lipo) were prepared for the temporally-controlled and multifaceted treatment of an advanced in vivo model of melanoma. Such combination therapy established strong synergistic effects, both in apoptotic extent and subsequent abrogation of tumor growth, which were due largely to both an enhanced cytotoxic T-cell recruitment and a reduction of immune-suppressive mediators in the microenvironments of both spleens and tumor. These results underlie a prolonged host lifespan in the combination approach (45 days) as compared with control (25 days, p < 0.02), providing promise toward a personalized approach to nanomedicine by establishing effect synergy in host-specific immunotherapy following chemotherapy.

Inflammasome-associated inflammation inhibits cell-mediated immune responses to Listeria monocytogenes

Abstract Listeria monocytogenes is a genetically tractable Gram-positive intracellular bacterium that activates a robust cell-mediated immune response capable of breaking self-tolerance. These properties contribute to its success as a cancer immunotherapeutic platform; however, despite these attributes, the specific mechanisms by which L. monocytogenes stimulates a robust adaptive immune response remain largely unknown. We hypothesized that cytosolic innate immune activation, specifically inflammasome activation, by L. monocytogenes would be critical for triggering a robust CD8+ T-cell response. Surprisingly, we found that inflammasome activation impairs the induction of antigen specific T-cells and ultimately the generation of protective immunity. To determine if this phenomenon is unique to inflammasome-induced pyroptosis or a general outcome of pathogen induced host cell death, we designed strains of L. monocytogenes to uniquely activate pyroptosis, necrosis, or apoptosis. Contrary to previous studies of immunity in the context of sterile cell death, pathogen induced pyroptosis, necrosis and apoptosis each impaired the generation of cell-mediated immunity. Importantly, the mechanism of impairment was unique for each form of cell death, although in each case it was independent of dendritic cell depletion or deficits in antigen presentation. Apoptosis and necrosis impaired dendritic cell co-stimulatory molecule expression, while the pyroptotic inflammatory milieu independently impaired the generation of cell-mediated immunity. These results suggest that inhibition of inflammasome activation, or specifically certain inflammatory components, is critical for the success of L. monocytogenes immunotherapy.

Induction of regulatory T-cells from memory T-cells is perturbed during acute exacerbation of multiple sclerosis

Regulatory T-cells (Tregs) are vital for maintaining immunological self-tolerance, and the transcription factor FOXP3 is considered critical for their development and function. Peripheral Treg induction may significantly contribute to the total Treg pool in healthy adults, and this pathway may be enhanced in thymic-deficient conditions like multiple sclerosis (MS). Here, we evaluated iTreg formation from memory versus naïve CD4+CD25− T-cell precursors. We report the novel finding that memory T-cells readily expressed CD25 and FOXP3, and demonstrated significantly greater suppressive function. Additionally, the CD25−FOXP3− fraction of stimulated memory T-cells also displayed robust suppression not observed in naïve counterparts or ex vivo resting (CD25−) T-cells. This regulatory population was present in both healthy subjects and clinically-quiescent MS patients, but was specifically deficient during disease exacerbation. These studies indicate that iTreg development and function are precursor dependent. Furthermore, MS quiescence appears to correlate with restoration of suppressive function in memory-derived CD4+CD25−FOXP3− iTregs.

Mutant KRAS Conversion of Conventional T Cells into Regulatory T Cells.

Constitutive activation of the KRAS oncogene in human malignancies is associated with aggressive tumor growth and poor prognosis. Similar to other oncogenes, KRAS acts in a cell-intrinsic manner to affect tumor growth or survival. However, we describe here a different, cell-extrinsic mechanism through which mutant KRAS contributes to tumor development. Tumor cells carrying mutated KRAS induced highly suppressive T cells, and silencing KRAS reversed this effect. Overexpression of the mutant KRAS(G12V)gene in wild-type KRAS tumor cells led to regulatory T-cell (Treg) induction. We also demonstrate that mutant KRAS induces the secretion of IL10 and transforming growth factor-β1 (both required for Treg induction) by tumor cells through the activation of the MEK-ERK-AP1 pathway. Finally, we report that inhibition of KRAS reduces the infiltration of Tregs in KRAS-driven lung tumorigenesis even before tumor formation. This cell-extrinsic mechanism allows tumor cells harboring a mutant KRAS oncogene to escape immune recognition. Thus, an oncogene can promote tumor progression independent of its transforming activity by increasing the number and function of Tregs. This has a significant clinical potential, in which targeting KRAS and its downstream signaling pathways could be used as powerful immune modulators in cancer immunotherapy.

Core Fucosylation on T Cells, Required for Activation of T-Cell Receptor Signaling and Induction of Colitis in Mice, Is Increased in Patients With Inflammatory Bowel Disease

Attachment of a fucose molecule to the innermost N-glycan in a glycoprotein (core fucosylation) regulates the activity of many growth factor receptors and adhesion molecules. The process is catalyzed by α1-6 fucosyltransferase (FUT8) and required for immune regulation, but it is not clear whether this process is dysregulated during disease pathogenesis. We investigated whether core fucosylation regulates T-cell activation and induction of colitis in mice, and is altered in patients with inflammatory bowel disease (IBD). Biopsy samples were collected from inflamed and noninflamed regions of intestine from patients (8 with Crohn's disease, 4 with ulcerative colitis, and 4 without IBD [controls]) at Osaka University Hospital. Colitis was induced in FUT8-deficient (Fut8(-/-)) mice and Fut8(+/+) littermates by administration of trinitrobenzene sulfonic acid. Intestinal tissues were collected and analyzed histologically. Immune cells were collected and analyzed by lectin flow cytometry, immunofluorescence, and reverse-transcription polymerase chain reaction, as well as for production of cytokines and levels of T-cell receptor (TCR) in lipid raft fractions. T-cell function was analyzed by intraperitoneal injection of CD4(+)CD62L(+) naïve T cells into RAG2-deficient mice. Levels of core fucosylation were increased on T cells from mice with colitis, compared with mice without colitis, as well as on inflamed mucosa from patients with IBD, compared with their noninflamed tissues or tissues from control patients. Fut8(-/-) mice developed less-severe colitis than Fut8(+/+) mice, and T cells from Fut8(-/-) miceproduced lower levels of T-helper 1 and 2 cytokines. Adoptive transfer of Fut8(-/-) T cells to RAG2-deficient mice reduced the severity of colitis. Compared with CD4(+) T cells from Fut8(+/+) mice, those from Fut8(-/-) mice expressed similar levels of TCR and CD28, but these proteins did not contain core fucosylation. TCR complexes formed on CD4(+) T cells from Fut8(-/-) mice did not signal properly after activation and were not transported to lipid rafts. Core fucosylation of the TCR is required for T-cell signaling and production of inflammatory cytokines and induction of colitis in mice. Levels of TCR core fucosylation areincreased on T cells from intestinal tissues of patients with IBD; this process might be blocked as a therapeutic strategy.

The successful induction of T-cell and antibody responses by a recombinant measles virus-vectored tetravalent dengue vaccine provides partial protection against dengue-2 infection

ABSTRACTDengue has a major impact on global public health, and the use of dengue vaccine is very limited. In this study, we evaluated the immunogenicity and protective efficacy of a dengue vaccine made from a recombinant measles virus (MV) that expresses envelope protein domain III (ED3) of dengue-1 to 4. Following immunization with the MV-vectored dengue vaccine, mice developed specific interferon-gamma and antibody responses against dengue virus and MV. Neutralizing antibodies against MV and dengue viruses were also induced, and protective levels of FRNT50 ≥ 10 to 4 serotypes of dengue viruses were detected in the MV-vectored dengue vaccine-immunized mice. In addition, specific interferon-gamma and antibody responses to dengue viruses were still induced by the MV-vectored dengue vaccine in mice that were pre-infected with MV. This finding suggests that the pre-existing immunity to MV did not block the initiation of immune responses. By contrast, mice that were pre-infected with dengue-3 exhibited no effect in terms of their antibody responses to MV and dengue viruses, but a dominant dengue-3-specific T-cell response was observed. After injection with dengue-2, a detectable but significantly lower viremia and a higher titer of anti-dengue-2 neutralizing antibodies were observed in MV-vectored dengue vaccine-immunized mice versus the vector control, suggesting that an anamnestic antibody response that provided partial protection against dengue-2 was elicited. Our results with regard to T-cell responses and the effect of pre-immunity to MV or dengue viruses provide clues for the future applications of an MV-vectored dengue vaccine.

Abstract B035: Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells

Abstract Background: Naturally occurring regulatory T (Treg) cells play an essential role for immunological unresponsiveness to self-tumor-antigens. Yet, how they stably suppress self-tumor-antigen-specific T cells remains unclear. In particular, it is unknown whether Treg cell-mediated suppression for a limited period has a critical long-lasting effect on cell fate and antigen reactivity of self-tumor-antigen-specific T cells. Experimental procedures: To demonstrate Treg-cell effect on proliferation, cytokine production, and cell fate of self-tumor-antigen-specific CD8+ T cells, CD8+ T cells from peripheral blood mononuclear cells (PBMCs) of healthy individuals were stimulated with a melanocyte differentiation antigen Melan-A/ MART-1, expressed by normal melanocytes and some melanoma cells, with/without FoxP3+CD25+CD4+ Treg cells. Melan-A/ MART-1-specific CD8+ T-cell induction and their phenotypes were investigated. Results: Melan-A/ MART-1-specific CD8+ T-cell induction was reduced by the presence of Treg cells. Self-tumor-antigen-specific CD8+ T cells co-cultured with Treg cells underwent one cell division and stopped further proliferation due to the lack of co-stimulation from antigen-presenting cells through Treg-cell suppression. They harbored low-affinity T-cell receptors and an anergic phenotype (i.e., hypo-proliferative and cytokine hypo-producing upon antigen re-stimulation) defined by expression of CCR7 and co-inhibitory molecules such as CTLA-4. Melan-A/ MART-1-specific CD8+ T cells with this anergic phenotype were detected ex vivo in the periphery and contributed to the tolerance of self-tumor-antigens. Conclusions: Treg-cell-mediated induction of anergy in self- antigen tumor-specific CD8+ T cells inhibits their activation in cancer patients after immunotherapy, and neo-antigens associated with tumor-specific gene mutations may become a main target of CD8+ T cells inducing actual tumor regression. Citation Format: Yuka Maeda, Hiroyoshi Nishikawa, Shimon Sakaguchi. Self-tumor-antigen-specific CD8 T cells were suppressed by regulatory T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B035.

Pathogenesis of minimal change nephrotic syndrome: an immunological concept.

Idiopathic nephrotic syndrome (INS) in children is characterized by massive proteinuria and hypoalbuminemia. Minimal change nephrotic syndrome (MCNS) is the most common form of INS in children. The pathogenesis of MCNS still remains unclear, however, several hypotheses have been recently proposed. For several decades, MCNS has been considered a T-cell disorder, which causes the impairment of the glomerular filtration barrier with the release of different circulating factors. Increased levels of several cytokines are also suggested. Recently, a "two-hit" theory was proposed that included the induction of CD80 (B7-1) and regulatory T-cell (Treg) dysfunction, with or without impaired autoregulatory functions of the podocyte. In contrast to the well-established involvement of T cells, the role of B cells has not been clearly identified. However, B-cell biology has recently gained more attention, because rituximab (a monoclonal antibody directed against CD20-bearing cells) demonstrated a very good therapeutic response in the treatment of childhood and adult MCNS. Here, we discuss recent insights into the pathogenesis of MCNS in children.