PBMC Of Healthy Donors Research Articles

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BackgroundCD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies.MethodsGBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401.ResultsGBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization.ConclusionThese results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Abstract P1-06-07: Immune-induced epithelial to mesenchymal transition in inflammatory breast cancer induces unique increases in E-cadherin, adhesion and migration through TNF-a, IL-6 and TGF-b

Abstract BACKGROUND AND RATIONALE Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and patients frequently present with metastases at the time of their diagnosis. Although a robust IBC-specific molecular signature remains elusive, the disease is frequently characterized by persistent expression of the adhesion molecule, E-cadherin. This is highly counterintuitive as epithelial to mesenchymal transition (EMT), frequently associated with metastasis, results in decreased E-cadherin expression and highly aggressive cancers frequently express low levels of E-cadherin. We hypothesized that persistent inflammation, mediated by immune activation, increases the plasticity of IBC cells, inducing EMT and allowing the re-acquisition of epithelia characteristics once removed from the inflammatory foci. In support of this hypothesis, previous in vitro work showed that soluble factors from activated immune cells induce EMT-related transcripts in both IBC and non-IBC cell lines. However, uniquely in 3 of 4 IBC cell lines but none of the non-IBC cell lines, this program included an increase of E-cadherin expression. RESULTS We used real-time cell analysis (RTCA) from Acea Biosciences (San Diego, CA) to probe the effect of immune conditioned media, produced by stimulating healthy donor peripheral blood mononuclear cells through the T-cell receptor or through toll-like receptor-4, on SUM149 inflammatory breast cancer cells. Consistent with the increased expression of E-cadherin, we observed rapid and strong increases in cellular adhesion as measured by the RCTA cell-index following culture with immune inflammatory factors. However, using the CIM chip, the same cells also showed strong increases in invasion and migration. To determine the inflammatory factors involved in this process, we screened the immune conditioned media using a Luminex array (Millipore, Billerica, MA). TGF-b, TNF-α, and IL-6, previously shown to induce EMT, were all found at elevated levels. In 5 culture supernatants of healthy donor PBMC activated for 48h with anti-CD3 antibody, TGF-β had a modest 1.6-fold increase; TNF-α had an average 101-fold increase; while IL-6 had an average 347-fold increase. When added to cultures of SUM149 cells, these factors recapitulated the EMT gene expression signature in SUM149 including the increase in E-cadherin expression. Furthermore, the addition of neutralizing antibodies against TNF-α, TGF-β, and IL-6 to immune conditioned media prior to exposure to SUM149 cells resulted in less EMT. CONCLUSIONS Inflammatory factors may induce both the migratory ability and the characteristic persistent E-cadherin expression of IBC cells. This is mediated in part by TNF-α, TGF-β, and IL-6. However, the molecular basis for this unique IBC response requires further study hindering the development of optimal therapies. Ongoing studies at MD Anderson are exploring both the tumor and stromal components of inflammatory breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-07.

From Bedside To Bench: Molecular Benchmarking Of An Fc-Optimized CD19 Antibody Used In Treatment Of Relapsed and Refractory Pediatric B-Lineage Acute Lymphoblastic Leukemia

B-lineage acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although this disease can be curatively treated in 80% of patients by chemotherapy, prognosis for primary refractory or relapsed patients is very poor. Even after allogeneic stem cell transplantation (SCT), relapse rates are considerable and correlate significantly with persistent minimal residual disease (MRD) prior to or after SCT. Since a MRD constellation represents favorable effector-target ratios it is well suited for immunotherapy with therapeutic antibodies.We developed and produced a third-generation CD19-specific monoclonal antibody (mAb) (4G7SDIE) in clinical-grade quality at a university-owned production unit. This high affinity Fc-optimized chimerized CD19-specific mAb mediates enhanced antibody-dependent cellular cytotoxicity (ADCC) by NK cells through its improved capability to recruit FcγRIIIa bearing effector cells. In this study, 4G7SDIE was applied within the scope of a compassionate use program in pediatric patients with relapsed or refractory B-lineage ALL and characterized in vitro and in vivo.Firstly, it was confirmed that CD19 is commonly and stably expressed in pediatric B-lineage ALL by quantitative flow cytometry analysis of primary leukemic blasts (mean expression: 1.4x104 CD19 molecules/cell; range 4.5x103-2.4x104; n = 18). Hence CD19 is a well suited target for immunotherapy of pediatric B-lineage ALL. Half-saturating concentrations of 4G7SDIE on primary leukemic blasts and cell line NALM-16 were reached at EC50= 85 ng/ml (± 29). Half-maximal target cell lysis was reached at EC50 = 25 ng/ml. Furthermore, lysis of primary B-lineage ALL blasts by PBMC of 4 healthy donors could be significantly increased by 22% when adding 1 µg/ml 4G7SDIE to donor serum in 2 h-cytotoxicity assays (n = 9; p = 0.03).4G7SDIE was applied in 11 pediatric patients with relapsed or refractory B-lineage ALL in order to reduce or eradicate MRD and thus prevent relapse in these high-risk pre- and post-transplant patients. Especially, in a post-transplant context, with a high number of allogeneic NK effector cells available, use of an ADCC-mediating mAb shows potential. In 6/9 treated patients with detectable MRD, leukemic load was reduced by ≥ 1 log or pushed below detection limit (10-4) through immunotherapy with 4G7SDIE. Moreover, 2 further patients responded to 4G7SDIE treatment. However, they received additional therapy with tyrosine-kinase inhibitors. Five of the treated patients eventually relapsed, 5 other patients went into remission after 4G7SDIE application (range 27-597 days).Concomitant in vitro 2 h-cytotoxicity assays with donor-derived PBMC of 2 treated patients showed that NK-cell mediated lysis of autologous B-lineage ALL blasts was increased by 33%, when adding 1 µg/ml 4G7SDIE or by 22% when adding autologous patient serum taken after antibody treatment (n = 8; p = 0.02). Serum half-life of 4G7SDIE in the first treatment cycles ranged between 20 h and 43 h and after infusion of 20 mg/m2, saturating serum concentrations of ≥700 ng/ml were detectable for at least 13 days.In a standardized model with MCF7-CD19-transfectants, expressing various CD19 levels on the cell surface, a correlation between increasing CD19 molecules/cell and increasing specific lysis by PBMC of healthy donors coincubated with 4G7SDIE was shown (spearman r = 0.88; p = 0.01). Strikingly, in 3 patients with residual disease detectable by flow cytometry, a down-modulation of CD19 on leukemic blasts under 4G7SDIE therapy was observed. In one patient up-regulation of CD19 after discontinuation of 4G7SDIE treatment was observed. In vitro antigenic shift assays on primary leukemic blasts showed considerable but very heterogeneous shift of CD19 surface expression. These observations hint at in vivotumor escape mechanisms and furthermore indicate selective pressure exerted by immunotherapy with 4G7SDIE, underlining its therapeutic potential, but also delineating its limitations.In conclusion, promising antileukemic effects have been observed in vitro and in vivo in this compassionate use program. However, potential CD19 down-modulation upon immunotherapy should be taken into account and may indicate the relevance of optimized treatment schedules and dosage as well as specific patient selection. We are currently setting up a clinical trial. Disclosures:No relevant conflicts of interest to declare.

Abstract 1272: Myeloid leukemia cell lines suppress T cell activation and proliferation.

Abstract In preliminary experiments to develop the K562 human leukemia cell line as an artificial antigen presenting cell we found that K562 cells strongly inhibited T cell proliferation. To explore this phenomenon further we tested CD4+CD25- T cells from healthy donor PBMC as targets of proliferation suppression. Targets were co-cultured with 3 chronic myeloid leukemia (CML) derived cell lines (K562, BV173 and CML1 established from a CML patient in our laboratory), two monocytic leukemia derived cell lines (THP-1 and U937), the myeloid leukemia derived cell line (KG1a) and the Burkitt lymphoma cell line (Daudi). To evaluate early activation, CD154 expression was tested by flow cytometry 16h after stimulation with CD3/CD28 beads. T cell proliferation was tested by CFSE dilution in T cells stimulated for 72h. All myeloid lines blocked CD154 expression (10.9-62.8% of control at 1:8 target: suppressor ratio). In CFSE proliferation assay, K562, KG1a, CML1, THP-1 and U937 induced comparable suppression (28.4-93.9%, at 1:8 ratio). In contrast, BV173 and Daudi cells were the least suppressive (4.96-23.0%, P<0.01). Strikingly, CML1 cell line and THP-1 showed more suppressive effect on T cell proliferation than CD4+CD25+CD127- autologous regulatory T cell (Treg). Our result showed that myeloid leukemia cell lines but not B cells strongly inhibit both T cell activation and T cell proliferation. Suppression of proliferation was greatest in acute myeloid and monocytic leukemia derived lines. These data indicate that myeloid lekemia cell lines exhibit features of myeloid-derived suppressor cells (MDSC) which can induce T cell tolerance to tumors. Our findings raise the possibility that myeloid leukemias may actively evade immune control through T cell suppression. We plan to use such cell lines to further define the mechanism of MDSC related T cell suppression. Citation Format: Kazushi Tanimoto, Sawa Ito, Samantha Miner, Pawel Muranski, Nancy F. Hensel, J. Joseph Melenhorst, A. John Barrett. Myeloid leukemia cell lines suppress T cell activation and proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1272. doi:10.1158/1538-7445.AM2013-1272

Abstract A41: Patterns and kinetics of interferon-γ secretion by natural killer cell subsets co-cultured with cetuximab-coated head and neck cancer cells and dendritic cells.

Abstract Despite over-expression of epidermal growth factor receptor (EGFR) in 80-90% of head and neck cancers (HNC), only ~20% of HNC patients are clinically responsive the EGFR-specific therapeutic monoclonal antibody (mAb) cetuximab. In addition to blocking EGFR signaling that leads to tumor cell survival and proliferation, increasing evidence suggests that innate and adaptive immunity likely plays a significant role in patient response to cetuximab. Natural killer (NK) cells recognize the Fc-region of EGFR-bound cetuximab via their activating Fcγ receptor IIIa (FcγRIIIa, also known as CD16) and mediate tumor cell lysis by antibody dependent cellular cytotoxicity (ADCC). NK cells are increasingly recognized as key mediators of crosstalk bridging the innate and adaptive arms of the immune response to human malignancies. We have previously shown that in addition to being direct effectors of ADCC against cetuximab-coated HNC cells, NK cells are instrumental in indirectly promoting an anti-tumor adaptive immune response via secretion of immunomodulatory cytokines such as IFNγ, which is known to enhance dendritic cell (DC) antigen processing and presentation to CD8+ T cells. Multiple studies have consistently demonstrated a relative decrease in the CD56dimCD16bright NK cell subset, traditionally considered to be primarily cytotoxic effectors, and an increase in the immunoregulatory CD56brightCD16dim NK cell subset in the peripheral blood of patients with various malignancies. However, given that the immunoregulatory CD56brightCD16dim NK cells are generally considered to be the more prolific cytokine producing subset, it was unclear how this redistribution of NK cell subsets might affect the dynamics of cytokine-mediated NK:DC crosstalk in the presence of mAb-coated tumor cells. Using primary NK cells and monocyte-derived DC isolated from PBMC of healthy donors, we therefore sought to better elucidate the patterns and kinetics of IFNγ secretion by the two NK cell subsets in response to cetuximab-coated HNC cells in the presence of DC. In this context, we found that while CD56brightCD16dim NK cells express more of the activating NKG2D receptor, rapid (by 4hrs) IFNγ secretion by CD56dimCD16bright NK cells is a CD16-dependent process. CD56brightCD16dim NK cells only produced IFNγ following stimulation for at least 12 hours with a combination of IL-12 and IL-15. IL-12/IL-15 stimulation also induced IFNγ secretion by CD56dimCD16bright NK cells, though to a lesser extent as compared to the CD56brightCD16dim subset. Taken together, these data contribute significantly to a mechanistic understanding of how NK cells function to bridge innate and adaptive anti-tumor responses in the context of mAb cancer therapies. Understanding this link is of broad relevance to the optimization of current therapeutic mAbs and will help devise novel ways to improve clinical responses. Citation Format: Christopher A. Lord, Raghvendra M. Srivastava, Robert L. Ferris. Patterns and kinetics of interferon-γ secretion by natural killer cell subsets co-cultured with cetuximab-coated head and neck cancer cells and dendritic cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A41.

P016 Impairment of TH1/TH17 host protective responses by macrophages alternative activation as a mechanism for helminthes survival

Human hydatidosis is caused by the larval stage of the metacestode Echinococcus granulosus (E.g.). It is one of the world’s major zoonotic infections. It usually manifests as unilocular cyst(s) mainly located in the liver. The prolonged coexistence of the parasite and its host involve the laminated layer (LL). This acellular layer represents an important barrier against immunological and physiological reactions of the host.We have previously highlighted an evident role of IFN γ in parasite killing by NOS2 (Nitric oxide Synthase2) pathway 1 . Moreover, IL-10/Arginase pathways seem to be implicated in parasite escape to host defense 2 . Moreover, LL impairs Th1/Th17 protective responses and allows the parasite to survive. This role may be mediated by Treg up-regulation by LL. In the present work, we aim to identify the early effect of LL in parasite establishment. In this sense, the effect of LL on macrophages phenotype and expression of some M1/M2 markers was investigated. NOS2 and Arginase activities were evaluated in cultures performed with PBMC of hydatid patients and healthy donors. Moreover, implication of mannose receptor (MR) and TGF β on Arginase activity was evaluated using mannose (MR antagonist) and Anti-TGF β . Activity of NADPH oxydase (ROS production) was also evaluated by chemiluminescence in cultures performed with human monocytes primed with NADPH oxydase agonists (like opsonized zymosan). Finally, TLR2, TLR4, CD14 and CD23 expression by the same cells was evaluated by flow cytometric immunoassay. We have found that NOS2 and NADPH activities are inhibited by LL. This effect is associated with Arginase activity stimulation. Interestingly, MR and TGF β seem to be implicated in the Arginase induction by LL. Moreover, LL increases TLR2 and CD14 expression and decreases TLR4 and CD23 expression by monocytes. Collectively, ours finding suggest that e.g. laminated layer induced M2 phenotype. These macrophages induce Treg cells and impair M1/Th1/Th17 protective responses allowing parasite survival. Inhibition of these mechanisms seems to be important issue to address during the design of anti-hydatic treatment.

Hyperthermia regulates FOXP3 expression in human FOXP3+ regulatory T cells (179.8)

Abstract RATIONALE: Fever is a primordial response to infection and inflammation. FOXP3+ regulatory T cells (Tregs) are central in orchestrating immune responses and preventing autoimmunity. This study hypothesizes that fever or hyperthermia regulates immunity by downregulating FOXP3 to abrogate thymic derived nTregs. METHODS: FACS analysis of FOXP3 in Tregs from peripheral blood of 5 adult cancer patients treated for 6h with thermal therapy (TT, 40 celcius). For in vitro studies, Tregs (CD4+CD127-CD25hi) were FACS-sorted from PBMC of healthy donors and stimulated with anti-CD3/CD28/IL2 for 6h at 37 (normothermia) or 40 (hyperthermia) celcius to assess their FOXP3 expression. Treg suppressive function was measured in a 4d CFSE based assay with daily 4h hyperthermia. RESULTS: Patients treated with TT had a significant (p<0.0001) decreased in geometric mean fluorescent intensity (gMFI) of FOXP3 concentration between 15-40% after 6h treatment that persisted after 18h later. There was a rise in the percentage of Tregs after 6h that fell below preheat after 18h postheat (p<0.001). In vitro studies demonstrated a decreased in the gMFI of FOXP3 in Tregs after 6h hyperthermia and an abrogation of suppressive function as compared to normothermia. CONCLUSIONS: Hyperthermia can regulate immune responses by transiently abrogating Treg suppressive function through the downregulation of FOXP3 expression. Hyperthermia has the therapeutic potential to augment vaccine response and cancer treatment.

CD11b/CD18 ligation on monocyte-derived dendritic cells dampens human Th17 cell responses (52.6)

Abstract Manipulating monocyte-derived DC (moDC) to alter effector T cell responses is an important strategy for the development of suppressive immunotherapy. We have previously shown that the ligation of CD11b/CD18 on moDC decreases the secretion of the Th17-skewing inflammatory cytokines IL-1β and IL-6, but maintains “immunosuppressive” TGF-β1. Here, we hypothesized that this “tolerogenic” DC-profile would diminish Th17 responses in peripheral human T cell populations. Healthy donor PBMC were differentiated into moDC in IL-4, GM CSF-supplemented media for 5 days. CD11b/CD18 on moDC was targeted via a bead-based surrogate system developed in our lab. Irrelevant IgG, or avb5, and beads without antibodies were used as controls. DC were stimulated with LPS and MDP or PGN or not stimulated, and then co-cultured with enriched CD4+ human T memory cells. IL-17 secretion was monitored per CBA after 24 hours and/or 4 days, and Th17 frequency assessed by ICS following restimulation with PMA/Ionomycin after 2 weeks. LPS/MDP or PGN-stimulated moDC induced significant increases in co-culture supernatant IL-17 levels and CD4+ Th17 cell frequencies (up to15%). Strikingly, these changes were significantly reduced in CD11b/CD18-ligated DC/T cell conditions, showing reversal of this NOD2/TLR4 or TLR2-mediated expansion effect though ligation of CD11b/CD18 on moDC.

HLA Anchor Optimization of the Melan-A–HLA-A2 Epitope within a Long Peptide Is Required for Efficient Cross-Priming of Human Tumor-Reactive T Cells

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16-40 LP bearing the HLA-A2-restricted epitope 26-35 or its analog 26-35(A27L) and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26-35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16-40(A27L) LP, for the treatment of HLA-A2(+) melanoma patients.

Identification and Characterization of Human Aspergillus Fumigatus-Specific Tr1-(Like) Cells

Abstract 181 Introduction:The ubiquitous mold Aspergillus fumigatus (A. fumigatus) induces two forms of pathogenesis: invasive aspergillosis in neutropenic patients and allergic aspergillosis in patients with chronic obstructive lung disease as well as in immunosuppressed patients. Mouse models of aspergillosis suggest that not only effector T cells (Teff) but also regulatory T cells (Treg) play a crucial role for the regulation of a protective T cell-mediated immunity to A. fumigatus. However, it is little-known about the involvement of Treg during A. fumigatus infection in humans. In order to develop new therapeutical strategies for the treatment of aspergillosis this project aims to understand the influence of regulatory T cells on A. fumigatus infection in humans.Material/Methods: A. fumigatus-specific CD4+ T cell clones were established from PBMC of healthy donors. Based on this clone pool Treg clones were identified due to their inability to proliferate in the absence of costimulation assessed by 3[H]-TdR incorporation as well as their Ag-specific cytokine production and phenotype determined by flow cytometry. Treg function was analyzed by their ability to suppress proliferation of autologous CD4+ T cells using CFSE dilution. Results:We identified A. fumigatus-specific T cell clones that exhibited marginal detectable proliferation after restimulation with immobilized anti-CD3 mAb in the absence of costimulation. However, these T cell clones vigorously proliferated in response to restimulation with their cognate antigen. A more detailed characterization showed that these suppressor T cell clones produced high amounts of IL-10 and moderate levels of IFN-gamma upon Ag-specific restimulation and expressed low amounts of Foxp3 but not Helios, a transcription factor that had recently been linked to natural occurring Treg. Most importantly, these T cell clones suppressed Ag-specific expansion of CD4+ Teff. This effect was contact-independent since suppression of Ag-specific CD4+ T cell expansion detected in transwell experiments was comparable to cocultures that enabled cellular-contact. Furthermore, anti-CD3/CD28-induced proliferation of naïve CD4+ T cells was not reduced in the presence of culture supernatants obtained from suppressor T cell clones after their antigen-specific restimulation in the absence of DCs. Conclusions:We identified for the first time A. fumigatus-specific CD4+ T cell clones with a Tr1(-like) IL-10+IFN-gamma+Foxp3lowHelios− phenotype. These cells suppressed expansion of A. fumigatus-specific Teff in an Ag-specific manner mediated by soluble factors released from Tr1(-like) cell clones. Since these factors did not affect CD4+ T cell proliferation in the absence of DCs our data suggest, that Tr1(-like) cell clones rather negatively regulate the stimulatory capacity of DCs leading to a reduced expansion of Ag-specific CD4+ T cells. Therefore these Tr1(-like) cells might play a protective role during A. fumigatus infection in humans. Thus, adoptive transfer of A. fumigatus-specific Treg could be useful to enhance protective immunity in patients with chronic A. fumigatus infection. Disclosures:Topp:Micromet: Consultancy, Honoraria.

Generation of Multi-Antigen Specific T Cells for Adoptive Immunotherapy of Myeloid Leukemia and Identification of MHC Class I and II-Restricted Peptides for WT1, Proteinase 3 and Human Neutrophil Elastase

Abstract 2985Generating cytotoxic T lymphocytes (CTL) against malignant cells using defined leukemia associated antigen (LAA) vaccines, or ex vivo adoptively transferred T cells, is a promising approach to prevent or treat leukemia. Vaccine or T cell therapies have largely focused on CD8 responses to defined HLA-A2 restricted LAA peptides. Strategies targeting single peptides are limited to the recipient’s HLA type, lack of CD4 help, and risk of tumor immune escape. To overcome these constraints and develop T cell immunotherapy for patients with myeloid malignancies, we used entire peptide libraries of several LAA to generate multi-specific CD4+ and CD8+ leukemia–reactive CTL. We obtained 15mer peptide libraries overlapping by 11 aa for WT1, Proteinase 3 (Pr3), MAGE-A3, PRAME and human neutrophil elastase (HNE). PBMC of 10 healthy donors were stimulated with DC pulsed with peptide libraries of one antigen or the mix of 5 LAA (LAA mix). Cultures were re-stimulated weekly with peptide-pulsed DCs and peptide-recognition was tested by IFN γ-ELIspot. Positive cultures were further characterized by performing intracellular cytokine staining, measuring degranulation by CD107a release, and assessing in vitro cytolytic activity using peptide-pulsed autologous PHA blasts as targets in a flow cytometric killing assay.Nine out of 10 healthy donors responded to the LAA mix as assessed by IFN γ-ELIspot (mean: 471.2 SFU/2×105 cells, range: 296–968.5). The expanded CTL specifically recognized 3 antigens (mean, range 1–5), and PRAME was most frequently recognized (in 9 of 10 donors screened). Within donors we consistently saw recognition of the same profile of antigens irrespective of whether the CTL were induced with individual antigen or LAA mix, indicating that antigenic competition between LAA was not a limiting factor in our cultures. Correlating with ELIspot results, LAA CTL were cytotoxic to PHAB pulsed with the LAA mix (E:T=40:1; mean: 33.1%, range: 12.4–80.0%; negative control: PHAB: mean: 4.2%, range: 0–13.4%; n=7), as well as individual antigens (WT1: mean :12.2%, range: 0–26.1%; Pr3: mean: 9.7%, range: 5.6–13.7%; HNE: mean: 35.4%, range: 0–84.9%; MAGE-A3: mean: 19.9%, range: 15.2–28.2%; PRAME: mean: 16.7%, range: 3.8–37.5%). Cytotoxicity against the peptide library or the LAA mix was greater than against targets pulsed with the immunodominant peptide, demonstrating the benefit of a broad immune response. Cytolytic activity was also seen in donors with predominantly CD4+ response, suggesting a role for T helper cells in tumor lysis.The approach of stimulation with LAA mix was also feasible for epitope mapping. In addition to known peptides, we identified 16 new MHC class I and II-restricted peptides (WT1: 8, HNE: 6 and Pr3: 2). Thereby, up to 4 immunogenic peptides of two different antigens could be identified in one LAA mix culture. WT1 peptides showed class I and class II-restriction by blocking experiments with HLA antibodies. HNE peptides were mainly class I-restricted, with a dominant response against RRLACLFLACVLPAL in 5 of 7 donors.Thus, stimulation of healthy donor PBMC with LAA mix generates CTL without depending on known HLA-restrictions and may be a practical approach for adoptive immunotherapy of myeloid leukemia after stem cell transplantation. Moreover, the strategy enabled the identification of several new class I and II-restricted peptides. Importantly, in vitro cytolytic activity could also be shown for MHC class II-restricted peptides, indicating the importance of CD4+ T cells for anti-leukemia responses. Disclosures:No relevant conflicts of interest to declare.

Lack of Th17 Cell Generation in Patients with Severe Burn Injuries

Immunodeficient patients with severe burn injuries are extremely susceptible to infection with Candida albicans. In addition to Th1 cells, IL-17-producing CD4(+) T cells (Th17 cells) have recently been described as an important effector cell in host anti-Candida resistance. In this study, therefore, we tried to induce Th17 cells in cultures of severely burned patient PBMC by stimulation with the C. albicans Ag (CAg). In the results, the biomarkers for Th17 cells (IL-17 production and intracellular expression of IL-17 and retinoic acid receptor-related orphan receptor γt) were not displayed by burn patient PBMC stimulated with CAg, whereas these biomarkers of Th17 cells were detected in cultures of healthy donor PBMC stimulated with CAg. Burn patient sera were shown to be inhibitory on CAg-stimulated Th17 cell generation in healthy donor PBMC cultures; however, Th17 cells were induced by CAg in healthy donor PBMC cultures supplemented with burn patient sera that were previously treated with anti-IL-10 mAb. Also, the biomarkers of Th17 cells were not induced by CAg in healthy donor PBMC cultures supplemented with rIL-10. IL-10 was detected in serum specimens derived from severely burned patients. These results indicate that Th17 cells are not generated in burn patient PBMC cultures supplemented with CAg. IL-10, produced in response to burn injuries, is shown to be inhibitory on Th17 cell generation. The high susceptibility of severely burned patients to C. albicans infection might be influenced if burn-associated IL-10 production is intervened.

Induction of antitumor immunity using dendritic cells electroporated with Polo-like kinase 1 (Plk1) mRNA in murine tumor models

Polo-like kinase 1 (Plk1), a serine-threonine kinase, plays a key role in the regulation of the cell cycle. Elevated Plk1 expression in various cancers is correlated with poor prognosis and poor patient survival rates. Several Plk1 inhibitors are currently being developed as potential treatments for cancer. In the present study, we investigated whether dendritic cells (DC) electroporated with mouse Plk1RNA (mPlk1RNA/DC) can induce Plk1-specific immune responses and exert antitumor effects in various murine tumor models. Overexpression of Plk1 protein was confirmed in several mouse and human tumor cell lines and various cancer tissues. Furthermore, Plk1-specific CD4(+) and CD8(+) T cells were induced by vaccination with mPlk1RNA/DC and the cytotoxic activity of the T cells was demonstrated against several Plk1-expressing tumor cell lines. Vaccination with mPlk1RNA/DC inhibited the growth of MC-38 and B16F10 tumors in C57BL/6 mice and the growth of CT26 tumors in BALB/c mice. Depletion of CD8(+) T cells reversed the inhibition of tumor growth by mPlk1RNA/DC vaccination. Homologous human Plk1RNA-electroporated DC also inhibited tumor growth in MC-38 tumor-bearing mice. In addition, Plk1-specific cytotoxic T lymphocytes from PBMC of healthy donors could be induced using autologous monocyte-derived DC electroporated with RNA encoding the whole gene of human Plk1. Taken together, the results of the present study suggest that Plk1 could be a universal tumor antigen recognized by cytotoxic T lymphocytes for cancer immunotherapy.

Functional characterization of human Cd33+ And Cd11b+ myeloid-derived suppressor cell subsets induced from peripheral blood mononuclear cells co-cultured with a diverse set of human tumor cell lines

BackgroundTumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSC). In cancer patients, MDSC accumulation correlates with increased tumor burden, but the mechanisms of MDSC induction remain poorly understood.MethodsThis study examined the ability of human tumor cell lines to induce MDSC from healthy donor PBMC using in vitro co-culture methods. These human MDSC were then characterized for morphology, phenotype, gene expression, and function.ResultsOf over 100 tumor cell lines examined, 45 generated canonical CD33+HLA-DRlowLineage- MDSC, with high frequency of induction by cervical, ovarian, colorectal, renal cell, and head and neck carcinoma cell lines. CD33+ MDSC could be induced by cancer cell lines from all tumor types with the notable exception of those derived from breast cancer (0/9, regardless of hormone and HER2 status). Upon further examination, these and others with infrequent CD33+ MDSC generation were found to induce a second subset characterized as CD11b+CD33lowHLA-DRlowLineage-. Gene and protein expression, antibody neutralization, and cytokine-induction studies determined that the induction of CD33+ MDSC depended upon over-expression of IL-1β, IL-6, TNFα, VEGF, and GM-CSF, while CD11b+ MDSC induction correlated with over-expression of FLT3L and TGFβ. Morphologically, both CD33+ and CD11b+ MDSC subsets appeared as immature myeloid cells and had significantly up-regulated expression of iNOS, NADPH oxidase, and arginase-1 genes. Furthermore, increased expression of transcription factors HIF1α, STAT3, and C/EBPβ distinguished MDSC from normal counterparts.ConclusionsThese studies demonstrate the universal nature of MDSC induction by human solid tumors and characterize two distinct MDSC subsets: CD33+HLA-DRlowHIF1α+/STAT3+ and CD11b+HLA-DRlowC/EBPβ+, which should enable the development of novel diagnostic and therapeutic reagents for cancer immunotherapy.

Transcriptomic biomarkers of the response of hospitalized geriatric patients admitted with heart failure. Comparison to hospitalized geriatric patients with infectious diseases or hip fracture

The abundance of a preselection of transcripts involved in inflammation, immunosenescence and stress response was compared between PBMC of healthy aged donors and aged patients in acute phase of heart failure and at recovery. This study identified 22 transcripts differentially abundant in acute phase of heart failure versus healthy aged subjects. Transcripts involved in inflammation and oxidative stress were more abundant. Those associated with T-cell functions were less abundant. The results were compared to two other major acute geriatric issues: infectious diseases and hip fracture. In acute phase, compared to healthy aged subjects, the abundance of 15/22 transcripts was also altered in both geriatric infectious diseases and hip fracture. Many variations had not vanished at the recovery phase. The abundance of CD28, CD69, LCK, HMOX1, TNFRSF1A transcripts, known to be altered in healthy aged versus healthy young subjects, was further affected in acute phase of the three geriatric diseases considered. The transcript levels of BCL2, CASP8, CCL5, DDIT3, EGR3, IL10RB, IL1R2, SERPINB2 and TIMP1 were affected in all three pathological conditions compared to healthy aged, but not versus healthy young subjects. In conclusion, this work provides critical targets for therapeutic research on geriatric heart failure, infectious diseases and hip fracture.

RANKL Expressed by Acute Myeloid Leukemia Cells Impairs NK Cell-Mediated Immune Surveillance

AbstractAbstract 2164NK cells play an important role in tumor immunosurveillance, especially of leukemia. Their reactivity is governed by various activating and inhibitory molecules expressed by their targets including multiple members of the TNF family. The TNF family member Receptor Activator of NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but recently have also been shown to influence progression of hematopoetic malignancies. Here we studied the yet unkown role of the RANK/RANKL molecule system in NK cells and their reactivity against acute myeloid leukemia (AML). Primary leukemia cells from AML patients were found to substantially express RANKL mRNA and surface protein in 75% of the investigated cases (n=40). Reverse signaling via surface-expressed RANKL into AML blasts induced the release of soluble factors including the immunoregulatory cytokines TNF and IL-10, which impaired NK cell anti-tumor reactivity. Moreover, we observed upregulation of RANK on NK cells among PBMC of healthy donors upon exposure to IL-10. This was not caused by direct effects on NK cells, but was rather due to yet unidentified factors released by monocytes among the PBMC upon IL-10 exposure and could be prevented by the activating cytokine IL-2. Furthermore, functional experiments with NK cells and RANKL transfectants or RANKL-negative controls revealed that forward signaling into RANK-expressing NK cells by tumor-expressed RANKL also directly impaired NK cytotoxicity and IFN-γ production. In line, blocking RANK-RANKL interaction using anti-RANKL antibodies or RANK-Fc fusion protein increased cytotoxicity and cytokine production of allogenic NK cells in cultures with RANKL-positive primary AML cells. Our data indicate that RANKL expression enables immune evasion of leukemia cells both by directly inhibiting reactivity of RANK-expressing NK cells and by orchestrating a reciprocal interplay between AML cells, monocytes and NK cells resulting in an immunosuppressive cytokine milieu. Thus, therapeutic modulation of the RANK/RANKL system, e.g. with Denosumab/AMG162, which is presently being evaluated for treatment of both non-malignant and malignant osteolysis, holds promise to reinforce NK reactivity against hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

4-1BB Ligand Mediates a “Vicious Cycle” of Immune Evasion Upon Reciprocal Interaction of Chronic Lymphocytic Leukemia and NK Cells

AbstractAbstract 2416NK cells play an important role in tumor immunosurveillance. Their reactivity is governed by various immunoregulatory molecules, which influence both direct anti-tumor immunity and NK responses induced by therapeutic antibodies like Rituximab. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells including NK cells. Recently we reported that the TNFR family member 4-1BB/CD137 is expressed on human NK cells following activation. In contrast to the stimulatory role of its murine counterpart, we found that human 4-1BB impairs NK anti-tumor reactivity upon interaction with its ligand 4-1BBL expressed on blasts of a substantial proportion of acute myeloid leukemia patients (Blood 115: 3058-69; 2010). In addition, we found that expression of 4-1BBL is general feature of leukemic cells of chronic lymphocytic leukemia (CLL) patients causing impaired direct and Rituximab-induced NK cell reactivity (Blood 114: 279; 2009). Here we report that reverse signaling via 4-1BBL into CLL cells following interaction with 4-1BB, which is absent on NK cells of healthy donors, but expressed at substantial levels on NK cells of CLL patients, induced pronounced production of immunoregulatory cytokines like TNF, IL-6 and IL-8 by the CLL cells. Moreover, we found that sera of CLL patients contained elevated levels of these immunoregulatory cytokines as compared to healthy controls. When PBMC of healthy donors were exposed to supernatants of in vitro cultured CLL cells or sera from CLL patients, this resulted in pronounced 4-1BB expression on the NK cells. This effect could be prevented by addition of the TNF blocker Infliximab to patient sera. The 4-1BB expression induced by CLL sera resulted in impaired NK reactivity specifically against 4-1BBL-expressing targets as revealed by functional analyses with 4-1BBL transfectants and the respective mock-controls. Moreover, the induced 4-1BB expression also impaired NK cell reactivity against primary CLL cells constitutively expressing 4-1BBL, thus closing a cycle of immune evasion. Taken together, our data demonstrate that 4-1BBL enables CLL cells to evade NK anti-tumor reactivity, and disruption of the “vicious 4-1BB-4-1BBL cycle” in CLL - NK interaction e.g. by TNF- or 4-1BB-blockade may serve well to enhance NK reactivity in therapeutic strategies like antibody treatment or allogenic stem cell transplantation in CLL, which rely on sufficient NK cell function. Disclosures:No relevant conflicts of interest to declare.

Efficient and Rapid In Vitro Generation of Multi-Virus-Specific CD4+ and CD8+ T Cells for Adoptive Immunotherapy.

AbstractAbstract 1175Adoptive transfer of T cells with specificity for one or several viruses is a promising strategy for the treatment or prevention of multiple opportunistic infections after allogenic stem cell transplantation. Here we have established a protocol for the generation of CMV, EBV and adenovirus (tri-virus) specific T cells. Virus-specific CD4+ and CD8+ T cells are stimulated in vitro using pools of peptides and subsequently magnetically enriched by virtue of their IFN-g secretion.The peptide pools are composed of synthetic peptides of mainly 15 amino acid length with 11 amino acid overlap covering the complete protein sequence of a viral antigen. We have analyzed the efficiency of short-term in vitro restimulation of cytomegalovirus (CMV)-specific CD4+ and CD8+ effector/memory T cells from CMV-infected healthy donors by CMV pp65 and IE-1 peptide pools as compared to single MHC class I restricted immunodominant pp65 and IE-1 9-mer peptides and CMV pp65 and IE-1 recombinant proteins based on flow cytometric analysis of the IFN-g response. The results show that peptide pools as well as recombinant proteins are useful for efficient activation of CD4+ T helper cells. In contrast, efficient stimulation of CD8+ T cells is achieved only using either the overlapping 15-mer peptide pools or the immunodominant 9-mer peptides.To test the usability of peptide pools to generate multi-virus-specific CD4+ and CD8+ T cells for adoptive immunotherapy we stimulated PBMC of several healthy donors with a combination of 5 peptide pools selected from CMV pp65, CMV IE-1, adenovirus hexon, EBV EBNA-1 and EBV BZLF-1 for 4 hours. Concomitant addition of 4 peptide pools to a single PBMC sample might decrease the activation efficiency for each peptide pool due to competition of peptides for MHC binding. Thus, as a control, we have divided PBMC into 4 aliquots, incubated each aliquot with a single peptide pool for 2 hours, and recombined the aliquots for T cell stimulation for another 4 hours. Afterwards, we used the IFN-g secretion assay technology to magnetically select for antigen-activated IFN-γ-secreting CD4+ and CD8+ T cells to a purity of > 90%. Antigen specificity and functionality of the enriched T cell population was examined after expansion. Within 9 – 14 days, T cells expanded between 4 and 745 fold. In addition, 21–53% of CD4+ T cells and 53–87% of CD8+ T cells re-expressed IFN-γ upon re-stimulation with the mixture of 4 peptide pools, confirming the high antigen specificity and functionality of the expanded T cells. Enrichment and expansions of T cells works equally well independent on whether the T cells are specific for CMV pp65, CMV IE-1, adenovirus hexon, EBV EBNA-1 or EBV BZLF-1. This can be deduced from the fact that the relative frequencies of T cells specific for each single antigen are about the same before and after the coenrichment and coexpansion process. For both approaches, the concomitant and separate antigen loading, we found comparable results with respect to recovery and purity of enriched IFN-γ-secreting T cells, expansion rate and antigen specificity of the expanded T cells.In summary, our results show that the combined usage of several peptide pools enables simultaneous and efficient activation of CMV, EBV and adenovirus (tri-virus) specific CD4+ and CD8+ T cells. Activated tri-virus specific T cells can be coenriched using the IFN-g secretion assay technology and used either directly or after in vitro coexpansion for adoptive immunotherapy. Disclosures:Richter:Miltenyi Biotec GmbH: Employment. Foerster:Miltenyi Biotec GmbH: Employment. Lasmanowicz:Miltenyi Biotec GmbH: Employment. Brauns:Miltenyi Biotec GmbH: Employment. Kramer:Miltenyi Biotec GmbH: Employment. Jekow:Miltenyi Biotec GmbH: Employment. Rönspeck:Miltenyi Biotec GmbH: Employment. Assenmacher:m: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.

A Comprehensive Ex Vivo Functional Analysis of Human NKT Cells Reveals Production of MIP1-α and MIP1-β, a Lack of IL-17, and a Th1-Bias in Males

NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-α, and MIP1-β as well as the Th1 cytokines IFN-γ and TNF-α. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-γ and MIP1-α, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases.

Apical TLR ligation of intestinal epithelial cells drives a T h1-polarized regulatory or inflammatory type effector response in vitro

Intestinal epithelial cells (IEC) separate the mucosal immune system from the external milieu. Under inflammatory conditions, Toll-like receptor (TLR) expression by IEC is increased. In a transwell co-culture model immune modulation by IEC upon TLR ligation was studied. Human IEC (HT-29 and T84) grown on filters were apically or basolaterally exposed to TLR4 or TLR9 ligands and co-cultured with CD3/CD28-activated healthy donor PBMC in the basolateral compartment. TLR4 ligation of IEC (HT-29) enhanced the production of TNF-α and IEC-derived MDC and decreased numbers of Foxp3 + regulatory T cells. Neutralization of TSLP abrogated TLR4-induced TNF-α secretion. In contrast, apical TLR9 ligation of IEC (HT-29 and T84) enhanced IFN-γ and IL-10 secretion and increased the number of activated T h1 cells. The increase in IFN-γ secretion depended on the presence of IEC. Furthermore, CD14 expression on monocytes was reduced coinciding with enhanced intracellular IL-10 and decreased TNF-α production. However, basolateral TLR9 ligand exposure of HT-29 cells resulted in enhanced IFN-γ, IL-6 and TNF-α, while IL-10 secretion remained unaltered. TLR4 and TLR9 ligands reduced IL-13 secretion in presence and absence of apically exposed IEC and enhanced IL-12 secretion in presence of IEC. These data suggest that TLR4 ligation of IEC drives an inflammatory, while apical TLR9 ligation drives a regulatory T h1 effector immune response in vitro in a polarized manner. IEC may be important modulators of the mucosal effector immune response.