CTL Effector Function Research Articles

Glycogen synthase kinase 3 controls T-cell exhaustion by regulating NFAT activation.

Cellular immunity mediated by CD8+ T cells plays an indispensable role in bacterial and viral clearanceand cancers. However, persistent antigen stimulation of CD8+ T cells leads to an exhausted or dysfunctional cellular state characterized by the loss of effector function and high expression of inhibitory receptors during chronic viral infection and in tumors. Numerous studies have shown that glycogen synthase kinase 3 (GSK3) controls the function and development of immune cells, but whether GSK3 affects CD8+ T cells is not clearly elucidated. Here, we demonstrate that mice with deletion of Gsk3α and Gsk3β in activated CD8+ T cells (DKO) exhibited decreased CTL differentiation and effector function during acute and chronic viral infection. In addition, DKO mice failed to control tumor growth due to the upregulated expression of inhibitory receptors and augmented T-cell exhaustion in tumor-infiltrating CD8+ T cells. Strikingly, anti-PD-1 immunotherapy substantially restored tumor rejection in DKO mice. Mechanistically, GSK3 regulates T-cell exhaustion by suppressing TCR-induced nuclear import of NFAT, thereby in turn dampening NFAT-mediated exhaustion-related gene expression, including TOX/TOX2 and PD-1. Thus, we uncovered the molecular mechanisms underlying GSK3 regulation of CTL differentiation and T-cell exhaustion in anti-tumor immune responses.

Metabolic Screening of Cytotoxic T-cell Effector Function Reveals the Role of CRAC Channels in Regulating Lethal Hit Delivery.

Cytotoxic T lymphocytes (CTL) mediate cytotoxicity toward tumor cells by multistep cell-cell interactions. However, the tumor microenvironment can metabolically perturb local CTL effector function. CTL activity is typically studied in two-dimensional (2D) liquid coculture, which is limited in recapitulating the mechanisms and efficacy of the multistep CTL effector response. We here developed a microscopy-based, automated three-dimensional (3D) interface coculture model suitable for medium-throughput screening to delineate the steps and CTL effector mechanisms affected by microenvironmental perturbation. CTL effector function was compromised by deregulated redox homeostasis, deficient mitochondrial respiration, as well as dysfunctional Ca2+ release-activated Ca2+ (CRAC) channels. Perturbation of CRAC channel function dampened calcium influx into CTLs, delayed CTL degranulation, and lowered the frequency of sublethal hits (i.e., additive cytotoxicity) delivered to the target cell. Thus, CRAC channel activity controls both individual contact efficacy and CTL cooperativity required for serial killing of target cells. The multistep analysis of CTL effector responses in 3D coculture will facilitate the identification of immune-suppressive mechanisms and guide the rational design of targeted intervention strategies to restore CTL effector function.

Abstract 1790: BO-112 as a modifier of the tumor microenvironment for liver metastases

Abstract Introduction: BO-112 is an analogue of double-stranded viral RNA which is an agonist of TLR3, RIG-1 and MDA5. It is currently in phase II clinical development, being administered intratumorally (IT), in combination with checkpoint inhibitors. It has been administered through different tumor locations, including liver metastases. Liver metastases constitute a challenging setting, with a proven negative impact on prognosis in those patients treated with immunotherapy independently of the tumor type but especially in those tumors considered as cold. Liver has a particular microenvironment, due to its continuous exposure to nonself-antigens. In order to deal with those antigens arising from the gut via the portal circulation, the liver must strike a balance between tolerating them and exhibiting some degree of antimicrobial effect. This microenvironment shows low level expression of antigen presenting cells, impaired to prime T cells. As a result, it is challenging to respond to exposed antigens, what may eventually prevent protective immune responses. A viral infection mimic induced by BO-112 results in upregulation of genes involved in T-cell homing and migration. This rationale provide support for the potential of IT BO-112 administration into liver metastases. Besides, PD-L1 induced upregulation in response to BO-112, suggests the need to combine treatment with an anti-PD-1/PD-L1 agent. Methods: BO-112 has been injected intratumorally into the liver in 16 patients so far at two different studies: a phase I trial (NCT02828098, which included initially patients with solid tumors being treated with BO-112 monotherapy and, through subsequent amendment, in combination with anti PD-1 drugs in patients having developed progressive disease on these therapies, being treated with BO-112 plus the same checkpoint inhibitor) and a phase II study (NCT04508140), which is including patients with liver metastases from colorectal (CRC) or gastroesophageal (GE) origin, receiving IT BO-112 plus pembrolizumab. A new phase II in patients with metastatic melanoma will start enrollment shortly and will also allow injection of liver metastases (NCT04570332). New unpublished data: Tumor biopsy results from phase 1 study (NCT02828098) showed increased expression of gene signatures for IFN, CD8 T-cell activation, CTL effector function and tumor inflammation. Changes in CD8 lymphocytes infiltration of the tumor microenvironment (TME) and PD-L1 expression in the injected liver metastases from CRC and GE tumors will be presented as part of an independent abstract sharing NCT04508140 preliminary results. Conclusions: Liver is a challenging organ to achieve clinical benefit in terms of response; however, with IT BO-112 being administered in liver metastases from different tumors, there is a change in the TME with an increase in key biomarkers, which may overcome primary or secondary resistance to systemic immunotherapy. Citation Format: Marisol Quintero, Vanesa Pons, Sonia Maciá, María Rojas, Javier Sánchez López, Desiree Kanters, Helena Escuin-Ordinas, Mark Branum, Juan M. Funes, Mercedes Pozuelo, Mike Doherty. BO-112 as a modifier of the tumor microenvironment for liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1790.

Optimized Dose of Dendritic Cell-based Vaccination in Experimental Model of Tumor Using Artificial Neural Network.

Previous studies have demonstrated that maturation of dendritic cells (DCs) by pathogenic components through pathogen-associated molecular patterns (PAMPs) such as Listeria monocytogenes lysate (LML) or CpG DNA can improve cancer vaccination in experimental models. In this study, a mathematical model based on an artificial neural network (ANN) was used to predict several patterns and dosage of matured DC administration for improved vaccination. The ANN model predicted that repeated co-injection of tumor antigen (TA)-loaded DCs matured with CpG (CpG-DC) and LML (List-DC) results in improved antitumor immune response as well as a reduction of immunosuppression in the tumor microenvironment. In the present study, we evaluated the ANN prediction accuracy about DC-based cancer vaccines pattern in the treatment of Wehi164 fibrosarcoma cancer-bearing mice. Our results showed that the administration of the DC vaccine according to ANN predicted pattern, leads to a decrease in the rate of tumor growth and size and augments CTL effector function. Furthermore, gene expression analysis confirmed an augmented immune response in the tumor microenvironment. Experimentations justified the validity of the ANN model forecast in the tumor growth and novel optimal dosage that led to more effective treatment.

P1‐005: Hepatitis B virus escapes non‐canonical CTL effector function

Journal of Viral HepatitisVolume 25, Issue S2 p. 36-37 Abstracts P1-005: Hepatitis B virus escapes non-canonical CTL effector function First published: 12 June 2018 https://doi.org/10.1111/jvh.04_12923Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume25, IssueS2Special Issue: 16th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 14th to 17th, 2018, in Toronto, CanadaJune 2018Pages 36-37 RelatedInformation

D-109 Defining a potent new class of latency reversing agents devoid of toxicity and detrimental cell activation that enhance CTL/NK cell killing

Despite long-term administration of antiretroviral therapy (ART), HIV-1 persists in a broadly distributed latent reservoir mainly comprised of resting CD4+ T cells. Cells harboring latent provirus typically display little to no HIV-1 gene expression and thus remain invisible to the immune system. To achieve a durable sustained viral remission in HIV-infected patients off ART (a functional cure), it will be important to both “reduce the size of the reservoir and to control viral rebound by eliciting an effective immune response capable of restraining viral spread from the smaller reservoir. This approach will likely require the combined use of potent and safe latency-reversing agent (LRA) and a therapeutic HIV vaccine. Our recent studies have shown that activators of the AKT/mTOR pathway form a promising group of LRAs. Our interest has focused on two small molecules, SB-216763 and Tideglusib that commonly inhibit glycogen synthase kinase-3 (GSK-3). This inhibition triggers a metabolic shift to glycolysis and results in sequential activation of mTORC2, AKT, and mTORC1. Both compounds are known to have high tissue penetration including the brain. We found that these GSK-3 inhibitors potently activate latent HIV-1 in both a tissue-based model of HIV latency formed in primary CD4 T cells, and in CD4 T cells isolated from HIV-infected patients. In some experiments, these agents are more potent than anti-CD3/anti-CD28 antibodies even though the GSK-3 inhibitors do not appear to induce T cell activation (measured by changes in CD69 and CD25 expression). Finally, in contrast to the undesirable compromise of CTL/NK function associated with many first generation LRAs (PKC activators, HDAC inhibitors), the GSK-3 inhibitors actually enhance CTL and NK cell effector function. In summary, our findings reveal GSK-3 inhibitors as an interesting new class of potent, safe, non-cell activating LRAs. Next, it will be key to define their activity in vivo using SIV-infected macaques on suppressive ART. The fact that tideglusib is already in phase II human trials for myotonic dystrophy and autism could accelerate its ultimate testing as an LRA in HIV-infected patients.

CD73 specific siRNA loaded chitosan lactate nanoparticles potentiate the antitumor effect of a dendritic cell vaccine in 4T1 breast cancer bearing mice

The efficacy of conventional anti-tumor immunotherapeutic approaches is markedly affected by the immunosuppressive microenvironment of tumor. Since adenosine is one of the main orchestra leaders in immunosuppression symphony of tumor, targeting its producing molecules such as CD73 can help to achieve a better clinical outcome following conventional cancer immunotherapeutic approaches. In the present study, we evaluated the efficacy of CD73-specific siRNA-loaded chitosan-lactate nanoparticles (ChLa NPs) in combination with tumor lysate pulsed dendritic cells (DCs) vaccine in treatment of 4T1 (murine derived) breast cancer bearing mice. Our results showed that intravenous administration of CD73-specific siRNA-loaded NPs led to reduced expression of CD73 in tumor cells which was associated with decreased tumor growth and metastasis, and improved mice survival. Furthermore, we found that the mechanism by which combination therapy inhibits tumor growth is in part related to downregulation of regulatory T (Treg), myeloid derived suppressor cells (MDSCs), and tumor associated macrophages, an augmented CTL effector function, improved proliferation status of T cells, increased production of inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17 and reduced levels of IL-10. Moreover, this treatment protocol attenuated the expression and activities of matrix metalloproteinases (MMPs) 2 and 9 which could be associated to the prevention of lung metastasis. In conclusion, our findings indicate that the use of CD73-specific siRNA-loaded NPs provides an immune potentiating function, thereby improves the efficacy of DC based cancer immunotherapy.

Calcium-dependence of cytotoxic granule endocytosis depends on the Flower protein

Abstract Cytotoxic T lymphocytes (CTLs) kill their targets by secreting perforin and granzymes from cytotoxic granules (CGs) at the interface between CTL and target cells, the immunological synapse (IS). Retrieval of membrane proteins at the IS plays a crucial role in CTL effector function by impacting the serial killing ability of CTLs. However, the cellular and molecular mechanism of CG endocytosis is largely unknown. In neuronal synapses, it has been shown that calcium influx is instrumental to trigger endocytosis, but the identity of calcium channel remains unclear. Flower protein has been reported as a potential candidate to regulate synaptic endocytosis in drosophila. We investigated a potential calcium-dependence of CG endocytosis and whether Flower plays a role in this process. We first confirmed that Flower protein is expressed in WT mouse CD8+ CTLs by western blot. We then generated Flower-deficient mice to further study Flower function. While we observed no change of degranulation and CG secretion in Flower-deficient CTLs, CG endocytosis at the IS was entirely blocked upon target cell contact. Re-introduction of Flower protein or elevation of extracellular calcium rescued the endocytosis defect in Flower-deficient CTLs. Interestingly, live imaging data showed that Flower localizes on intracellular vesicles which polarize to the IS upon target cell contact. We conclude from our data that endocytosis of CGs is calcium-dependent and that the Flower protein is providing the required calcium, probably through release from intracellular organelles.

CTL-Promoting Effects of IL-21 Counteract Murine Lupus in the Parent→F1 Graft-versus-Host Disease Model.

IL-21 promotes B cell and CTL responses in vivo, conferring IL-21 with a role in both humoral and cellular responses. Because CTL can target and eliminate autoreactive B cells, we investigated whether IL-21R signaling in CD8 T cells would alter the expansion of autoreactive B cells in an autoimmune setting. We addressed this question using the parent→F1 murine model of acute and chronic (lupus-like) graft-versus-host disease (GVHD) as models of a CTL-mediated or T-dependent B cell-mediated response, respectively. Induction of acute GVHD using IL-21R-deficient donor T cells resulted in decreased peak donor CD8 T cell numbers and decreased CTL effector function due to impaired granzyme B/perforin and Fas/Fas ligand pathways and a phenotype of low-intensity chronic GVHD with persistent host B cells, autoantibody production, and mild lupus-like renal disease. CTL effector maturation was critically dependent on IL-21R signaling in Ag-specific donor CD8, but not CD4, T cells. Conversely, treatment of DBA/2J→F1 chronic GVHD mice with IL-21 strongly promoted donor CD8 T cell expansion and rescued defective donor anti-host CTLs, resulting in host B cell elimination, decreased autoantibody levels, and attenuated renal disease, despite evidence of concurrently enhanced CD4 help for B cells and heightened B cell activation. These results demonstrate that, in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, IL-21 signaling on Ag-specific donor CD8 T cells is critical for CTL effector maturation, whereas a lack of IL-21R downregulates CTL responses that would otherwise limit B cell hyperactivity and autoantibody production.

High Avidity CD8+ T Cells Efficiently Eliminate Motile HIV-Infected Targets and Execute a Locally Focused Program of Anti-Viral Function

The dissemination of HIV from an initial site of infection is facilitated by motile HIV-infected CD4+ T-cells. However, the impact of infected target cell migration on antigen recognition by HIV-specific CD8+ T-cells is unclear. Using a 3D in vitro model of tissue, we visualized dynamic interactions between HIV-infected or peptide-pulsed CD4+ T-cells and HIV-specific CD8+ T-cells. CTLs engaged motile HIV-infected targets, but ∼50% of targets broke contact and escaped. In contrast, immobilized target cells were readily killed, indicating target motility directly inhibits CD8+ T-cell function. Strong calcium signals occurred in CTLs killing a motile target but calcium signaling was weak or absent in CTLs which permitted target escape. Neutralization of adhesion receptors LFA-1 and CD58 inhibited CD8+ T-cell function within the 3D matrix, demonstrating that efficient motile target lysis as dependent on adhesive engagement of targets. Antigen sensitivity (a convolution of antigen density, TCR avidity and CD8 coreceptor binding) is also critical for target recognition. We modulated this parameter (known as functional avidity but referred to here as “avidity” for the sake of simplicity) by exploiting common HIV escape mutations and measured their impact on CTL function at the single-cell level. Targets pulsed with low avidity mutant antigens frequently escaped while CTLs killed targets bearing high avidity antigen with near-perfect efficiency. CTLs engaged, arrested, and killed an initial target bearing high avidity antigen within minutes, but serial killing was surprisingly rare. CD8 cells remained committed to their initial dead target for hours, accumulating TCR signals that sustained secretion of soluble antiviral factors. These data indicate that high-avidity CD8+ T-cells execute an antiviral program in the precise location where antigen has been sensed: CTL effector functions are spatiotemporally coordinated with an early lytic phase followed by a sustained stationary secretory phase to control local viral infection.

CD103 or LFA-1 Engagement at the Immune Synapse between Cytotoxic T Cells and Tumor Cells Promotes Maturation and Regulates T-cell Effector Functions

T-cell adhesion/costimulatory molecules and their cognate receptors on target cells play a major role in T-cell receptor (TCR)-mediated activities. Here, we compared the involvement of CD103 and LFA-1, and their respective ligands, in the maturation of the cytotoxic immune synapse (cIS) and in the activation of CTL effector functions. Our results indicate that cytotoxicity toward cancer cells and, to a lesser extent, cytokine production by specific CTL require, together with TCR engagement, the interaction of either CD103 with E-cadherin or LFA-1 with ICAM-1. Flow-based adhesion assay showed that engagement of CD103 or LFA-1, together with TCR, enhances the strength of the T-cell/target cell interaction. Moreover, electron microscopic analyses showed that integrin-dependent mature cIS (mcIS) displays a cohesive ultrastructure, with tight membrane contacts separated by extensive clefts. In contrast, immature cIS (icIS), which is unable to trigger target cell lysis, is loose, with multiple protrusions in the effector cell membrane. Experiments using confocal microscopy revealed polarized cytokine release and degranulation at the mcIS associated with target cell killing, whereas icIS is characterized by failure of IFN-γ and granzyme B relocalization. Thus, interactive forces between CTL and epithelial tumor cells, mainly regulated by integrin engagement, correlate with maturity and the ultrastructure of the cIS and influence CTL effector functions. These results provide new insights into molecular mechanisms regulating antitumor CTL responses and may lead to the development of more efficient cancer immunotherapy strategies.

ADAM10 CONTRIBUTES TO MICA/B SHEDDING FROM MULTIPLE MYELOMA CELLS IN RESPONSE TO GENOTOXIC STRESS

Event Abstract Back to Event ADAM10 CONTRIBUTES TO MICA/B SHEDDING FROM MULTIPLE MYELOMA CELLS IN RESPONSE TO GENOTOXIC STRESS Francesca Cecere1*, Alessandra Zingoni1*, Elisabetta Vulpis1, Rosa Molfetta1, Alessandra Soriani1, Cinzia Fionda1, Maria Teresa Petrucci2, Maria Rosaria Ricciardi2, Rossella Paolini1, Marco Cippitelli1 and Angela Santoni1 1 "Sapienza" University of Rome, Molecular Medicine, Italy 2 "Sapienza" University of Rome, Hematology, Italy The MHC class I-related chain MICA/B ligands for the activating receptor NKG2D can be shed from tumor cells and their presence in sera is associated with impaired NK and CTL effector functions and disease progression. We showed that multiple myeloma (MM) cells up-regulate surface expression of NKG2D ligands after treatment with low doses of chemotherapeutic drugs and become more susceptible to NK cell lysis. Here, we investigated whether drug treatment of MM cells could result in MICA/B release. Soluble MICB was up-regulated after drug treatment and the ADAM10 specific inhibitor GI254023X dramatically reduced MICB shedding in a dose-dependent manner and was accompanied by a concomitant increase of cell-surface MICB. Similar results were obtained by shRNA-mediated gene silencing. Remarkably, chemotherapeutic agents increased ADAM10 expression either in MM cell lines or in ex-vivo malignant CD138+/CD38+ plasma cells. Regarding MICA, since its polymorphism can affect the shedding process, we investigated the effect of chemotherapeutic agents on the release of two allelic variants of MICA: MICA*019 and MICA*008 which represent the prototype of the long and short form of the MICA alleles, respectively. Similarly to MICB, drug treatment stimulated MICA*019 shedding with an ADAM10 dependent mechanism while didn’t affect MICA*008 release. Our findings indicate that chemotherapeutic drugs not only enhance MICA/B surface expression on MM cells, but also promote their shedding. The MICA genotype and the relative contribution of membrane associated vs soluble MICA/B in NKG2D dependent mechanisms of NK cell recognition of MM cells, should be considered to design NK cell-based immunochemotherapeutic approaches. References Soriani, A., Zingoni, A., Cerboni, C., Iannitto, M.L., Ricciardi, M.R., Di Gialleonardo, V., Cippitelli, M., Fionda, C., Petrucci, M.T., Guarini, A., Foa’, R., Santoni, A. (2009). ATM-ATR dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype. Blood, 113, 3503-3511. Keywords: Natural Killer cells, NKG2D, Metalloproteinases, Multiple Myeloma, MICA polymorphism Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Cecere F, Zingoni A, Vulpis E, Molfetta R, Soriani A, Fionda C, Petrucci M, Ricciardi M, Paolini R, Cippitelli M and Santoni A (2013). ADAM10 CONTRIBUTES TO MICA/B SHEDDING FROM MULTIPLE MYELOMA CELLS IN RESPONSE TO GENOTOXIC STRESS. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00115 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: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Francesca Cecere, "Sapienza" University of Rome, Molecular Medicine, Rome, Italy, francesca.cecere@uniroma1.it Dr. Alessandra Zingoni, "Sapienza" University of Rome, Molecular Medicine, Rome, Italy, alessandra.zingoni@uniroma1.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni Google Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni Google Scholar Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni PubMed Francesca Cecere Alessandra Zingoni Elisabetta Vulpis Rosa Molfetta Alessandra Soriani Cinzia Fionda Maria Teresa Petrucci Maria Rosaria Ricciardi Rossella Paolini Marco Cippitelli Angela Santoni Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Induction of antigen-specific human T suppressor cells by membrane and soluble ILT3

Antigen-specific CD8 suppressor T cells (CD8+ Ts) are adaptive regulatory T cells that are induced in vivo and in vitro by chronic antigenic stimulation of human T cells. CD8+ Ts induce the upregulation of the inhibitory receptors ILT3 and ILT4 on monocytes and dendritic cells rendering these antigen presenting cells (APCs) tolerogenic. Tolerogenic APCs induce CD4+ T helper anergy and elicit the differentiation of CD4+ and CD8+ T regulatory/suppressor cells. Overexpression of membrane ILT3 in APC results in inhibition of NF-κB activation, transcription of inflammatory cytokines and costimulatory molecules. Soluble ILT3-Fc which contains only the extracellular, Ig-like domain linked to mutated IgG1 Fc, is strongly immunosuppressive. ILT3-Fc, induces the differentiation of human CD8+ Ts which inhibit CD4+ Th and CD8+ CTL effector function both in vitro and in vivo. The acquisition of Ts' function by primed CD8+ T cells treated with ILT3-Fc was demonstrated to be the effect of the significant upregulation of BCL6, a transcriptional repressor of IL-2, IFN-gamma, IL-5 and granzyme B. The upregulated expression of BCL6, SOCS1 and DUSP10 is integral to the signature of ILT3-Fc-induced CD8+ Ts. These genes are known inhibitors of cytokine production and TCR signaling and are targeted by miRNAs which are suppressed by ILT3-Fc. ILT3-Fc induces tolerance to allogeneic human islets and reverses rejection after its onset in a humanized NOD/SCID mouse model. Based on these findings we postulate that ILT3-Fc may become an important new agent for treatment of autoimmunity and transplant rejection.

Affinity Thresholds for Naive CD8+ CTL Activation by Peptides and Engineered Influenza A Viruses

High-avidity interactions between TCRs and peptide + class I MHC (pMHCI) epitopes drive CTL activation and expansion. Intriguing questions remain concerning the constraints determining optimal TCR/pMHCI binding. The present analysis uses the TCR transgenic OT-I model to assess how varying profiles of TCR/pMHCI avidity influence naive CTL proliferation and the acquisition of effector function following exposure to the cognate H-2K(b)/OVA(257-264) (SIINFEKL) epitope and to mutants provided as peptide or in engineered influenza A viruses. Stimulating naive OT-I CD8(+) T cells in vitro with SIINFEKL induced full CTL proliferation and differentiation that was largely independent of any need for costimulation. By contrast, in vitro activation with the low-affinity EIINFEKL or SIIGFEKL ligands depended on the provision of IL-2 and other costimulatory signals. Importantly, although they did generate potent endogenous responses, infection of mice with influenza A viruses expressing these same OVA(257) variants failed to induce the activation of adoptively transferred naive OT-I CTLps, an effect that was only partially overcome by priming with a lipopeptide vaccine. Subsequent structural and biophysical analysis of H2-K(b)OVA(257), H2-K(b)E1, and H2-K(b)G4 established that these variations introduce small changes at the pMHCI interface and decrease epitope stability in ways that would likely impact cell surface presentation and recognition. Overall, it seems that there is an activation threshold for naive CTLps, that minimal alterations in peptide sequence can have profound effects, and that the antigenic requirements for the in vitro and in vivo induction of CTL proliferation and effector function differ substantially.

Abstract 473: CD4+ T cell help differentially influences anti-tumor immunity as a function of T cell avidity

Abstract One of the major hurdles for cancer immunotherapy is that most tumors express self-antigens to which the immune system is unresponsive due to self-tolerance. Our lab and others have demonstrated that high-avidity tumor-specific CD8+ T cells are superior in anti-tumor activity to low avidity T cells. However, adoptive transfer of high-avidity, tumor-specific CTL populations may fail due to loss of CTL effector functions upon tumor infiltration. Therefore, strategies to maintain effector functions of high avidity CTL, or enhancement of low avidity CTL are desirable. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high-avidity CTLs in anti-tumor immunity as compared to lower avidity T cells, we utilized two transgenic mouse lines developed in our lab that bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 TCR Tg mice (TCRhi) show higher avidity than those from clone 37 (TCRlo) mice based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells (OVA323-339 specific, CD4-OVA) with TRP-2-specific CD8+ T cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-γ secretion, Gr-B secretion as well as cell-mediated cytotoxicity, but had minimal effect on TCRlo cells. We observed that only adoptive co-transfer of CD4-OVA cells with TCRhi cells suppressed tumor growth of (5 day) established B16 melanoma tumors. Furthermore, those mice receiving co-transferred CD4-OVA cells with TCRhi cells developed autoimmune depigmentation at the sites of vaccination and tumor challenge. Our finding may have important implications for enhancement of cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 473. doi:10.1158/1538-7445.AM2011-473

FCRL6 receptor: Expression and associated proteins

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63 kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.

Provision of CD4+ T cell help preferentially enhances anti-tumor immunity of high avidity T cells (131.19)

Abstract It has been shown that high avidity tumor-specific CD8+ T cells are superior to low-avidity T cells in anti-tumor activity. However, adoptive transfer of high-avidity CTL populations could still fail as a therapy due to the fact that CTL effector function may be lost following tumor infiltration. To investigate whether provision of CD4+ T cell help will enhance the effectiveness of high avidity CTLs in anti-tumor immunity, we utilized transgenic mice developed in our lab which bear different TCR transgenes specific for a common melanoma differentiation antigen, tyrosinase-related protein 2 (TRP-2(180-188)). T cells from clone 24 Tg mice (TCRhi) show higher avidity than those from clone 37 Tg mice (TCRlo) based on tetramer dissociation and in vitro effector function assays. Here we report that co-activation of CD4+ T cells with TCRhi cells can boost the in vitro function of TCRhi cells, including proliferation, IFN-gamma secretion, Gr-B secretion, and antigen-specific cytotoxicity, but had minimal effects on TCRlo cells. In the treatment of subcutaneous B16 melanoma, we observed that adoptive co-transfer of CD4+ T cells with TCRhi cells (not with TCRlo cells) markedly suppressed tumor growth and induced autoimmune depigmentation at the sites of vaccination and tumor challenge. These findings suggest that provision of CD4+ T cell help preferentially enhances anti-tumor immunity of high-avidity T cells, which may have critical implications for cancer therapy.

Loss of DNAM‐1 contributes to CD8+ T‐cell exhaustion in chronic HIV‐1 infection

The hallmark of chronic viral infections is a progressive exhaustion of antigen-specific CD8(+) T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8(+) T cells that makes them dysfunctional. Here, we show that during human chronic HIV-1 infection, a CD8(+) T-cell positive costimulatory pathway mediated by DNAX-activating molecule-1 is also disrupted. Thus, DNAX-activating molecule-1 downregulation on CD8(+) T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection.

The duration of TCR/pMHC interactions regulates CTL effector function and tumor‐killing capacity

Effector CTL contribute to tumoral immunity by killing tumor cells through secretion of cytotoxic granules and cytokines. Activation of CTL requires specific recognition of cognate peptide-MHC-I (pMHC) complexes on the tumor cell surface by the CTL TCR. It has been suggested that the half-life (t(1/2)) of the TCR/pMHC interaction modulates the activation of naïve CD8(+) T cells; however, it remains unknown whether CTL effector function can also be regulated by the TCR/pMHC t(1/2). Here, we have studied CTL activity in response to tumor cells loaded with pMHC that bind the TCR with different t(1/2). We observed that the TCR/pMHC t(1/2) can differentially regulate CTL effector function during the interaction with tumor cells and defines the nature of anti-tumoral CTL responses in vivo. Although prolonged TCR/pMHC t(1/2) promoted only partial expression of cytotoxic molecules, short t(1/2) induced partial polarization of lytic machinery toward target cells. In contrast, intermediate TCR/pMHC t(1/2) induced strong expression of cytotoxic molecules, efficient polarization of lytic machinery and subsequent release of toxic granules by CTL that killed tumor cells. Consistently, efficient in vivo CTL-mediated tumor clearance was only observed for tumors expressing intermediate t(1/2) pMHC ligands. These data suggest that there is an optimal TCR/pMHC t(1/2) for efficient CTL activity.

Involvement of CD252 (CD134L) and IL-2 in the Expression of Cytotoxic Proteins in Bacterial- or Viral-Activated Human T Cells

Regulation of cytotoxic effector molecule expression in human CTLs after viral or bacterial activation is poorly understood. By using human autologous dendritic cells (DCs) to prime T lymphocytes, we found perforin only highly up-regulated in virus- (HSV-1, vaccinia virus) but not in intracellular bacteria- (Listeria innocua, Listeria monocytogenes, Mycobacterium tuberculosis, Chlamydophila pneumoniae) activated CTLs. In contrast, larger quantities of IFN-gamma and TNF-alpha were produced in Listeria-stimulated cultures. Granzyme B and granulysin were similarly up-regulated by all tested viruses and intracellular bacteria. DCs infected with HSV-1 showed enhanced surface expression of the costimulatory molecule CD252 (CD134L) compared with Listeria-infected DC and induced enhanced secretion of IL-2. Adding blocking CD134 or neutralizing IL-2 Abs during T cell activation reduced the HSV-dependent up-regulation of perforin. These data indicate a distinct CTL effector function in response to intracellular pathogens triggered via differing endogenous IL-2 production upon costimulation through CD252.