IFN-gamma Responses Research Articles

Abstract B33: Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model

Abstract Objective: Therapeutic success currently seen with immune checkpoint blockade (ICB) occurs primarily in immunogenic tumors and can be partly explained by the high rates of non-synonymous genetic mutations (single nucleotide variants, SNV) in tumor cells, leading to neoepitope presentations and robust T-cell infiltration. Despite showing mutations in fewer genes (most frequently TP53), high-grade serous ovarian cancer (HGSOC) displays signs of T-cell inflammation and responds, albeit modestly, to ICB. We focus on immune therapy tailored to the genomic characteristics of the tumor, especially to structural aberrations (SAs), like gene fusions. Our hypothesis is that SAs that are expressed as proteins are antigenic and immunogenic, and represent a source of neoantigens for combination immunotherapies that can overcome locoregional immune suppression. DNA structural aberrations can result in intergenic fusions and intragenic rearrangements, such as exon duplications (dup) and deletions (del). Here we focus on identifying gene fusion and exon dup/del-derived neoepitopes based on RNA-seq data for a combination of retrospective and prospective sample collections. Our hypothesis is that these gene rearrangements resulting from SVs may contribute to the immunogenicity and antigenicity of HGSOC. Methods: FusionCatcher was used to evaluate RNA sequencing data for in-frame gene fusions events in banked human HGSOC. HLAminer was used to determine individual haplotypes. Upon detection of fusion events (validated via PCR) we identified the MHC-I and MHC-II restricted peptides with predicted high or intermediate (IC50<500nM) binding affinity. The peptides (with sequences across the fusion point) were synthesized and pulsed onto HLA-matched PBMCs, for in vitro T cells stimulation assay. Following antigen priming and in vitro restimulation, IFNγ production by T cells was measured by ELISA. The efficacy of SA-derived neoepitopes was tested in vivo with two new preclinical mouse models based on MKP-Lung and MKP-Liver cells ovarian cancer cell lines. RNA-seq data of their complete mutanome were used to generate neoepitopes. RNA from normal mouse OSE isolated from healthy mice was used as reference for mutation calling. For vaccine generation, peptides were pulsed onto dendritic cells matured into Th1-inducing phenotype (DC1). Mice were treated with Rat IgG, anti-PDL1, or vaccine + anti PD-L1. Objective response was measured by tumor volume. Results: Our results demonstrate neoepitopes-specific human T-cell responses in vitro. RNA-seq data were available for nine patients; eight MHC-I and five MHC-II neoantigens were synthesized from gene fusions from three different patients represented by six sets of haplotype specific PBMCs. In vitro, neoantigen-treated PBMCs showed increased cell clumping. An objective IFN gamma response on ELISA was seen for six peptides at two different concentrations and five peptides at one concentration. Analysis of RNA-seq of murine tumors shows that the metastatic MKP-Lung cells (which carry molecular signatures associated with recurrent ovarian cancer), have an increased number of mutations (SNVs, indels) including gene fusions, in line with findings from human tumors. Using in silico prediction tools, we identified 16 potential peptide binders (IC50< 500), two with strong and 14 with moderate binding. The transcript variants were validated via PCR. The vaccine comprised the top seven MHC-I restricted peptide candidates with IC50<500Nm and one large (MHC-II) candidate peptide for CD4 T-cell responses. No difference was seen between treatment groups (ANOVA p=0.35). Conclusion: Our results demonstrate that PBMC pulsed with a combination of short and large peptides release increased IFNγ, supporting the proposed vaccine design. Structural variants contribute to tumor immunogenic potential. Immunogenicity data from this project will be further used to refine the target selection for individualized, mutanome-based vaccines. Citation Format: Malcolm S. Ross, Ma Tianzhou, Lixin Zhang, Nolan Priedigkeit, George Tseng, Adrian V. Lee, Robert P. Edwards, Anda M. Vlad. Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B33.

Expansion of tumor infiltrating lymphocytes (TIL) from bladder cancer

ABSTRACTAdvanced bladder cancer patients have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has been used successfully in treating patients with metastatic melanoma, resulting in a median OS of 52 months. In this study, we investigated the feasibility of expanding TIL from the tumors of bladder cancer patients. Primary bladder tumors and lymph node (LN) metastases were collected. Tumor specimens were minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. Expanded TIL were phenotyped by flow cytometry and anti-tumor reactivity was assessed after co-culture with autologous tumor digest and IFN-gamma ELISA. Of the 28 transitional cell bladder or LN tumors collected, 14/20 (70%) primary tumors and all of the LN metastases demonstrated TIL expansion. Expanded TIL were predominantly CD3+ (median 63%, range 10–87%) with a median of 30% CD8 + T cells (range 5–70%). TIL secreted IFN-gamma in response to autologous tumor. Addition of agonisitic 4-1BB antibody improved TIL expansion from primary bladder tumors regardless of pre-treatment with chemotherapy. This study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer.

Abstract 1024: Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples

Abstract Introduction: Anti-tumor immune response is controlled by a complex interaction between the immune system, tumor cells, and associated stroma. Expression of major histocompatibility complex class I (MHC I) and MHC class II (MHC II) antigens may be dysregulated in cancer, leading to alterations in the tumor antigen presentation profile. Stage I–III resected NSCLC tumor samples were profiled to study the relationship between MHC I, MHC II, and programmed death ligand 1 (PD-L1) tumor cell (TC) expression with tumor-associated inflammation. Here we report the potential impact of respective profiles on tumor immune response. Methods: 53 adenocarcinoma (NSCLC-AD) and 51 squamous cell carcinoma (NSCLC-SQ) stage I–III resected formalin-fixed, paraffin-embedded tumor specimens from commercial sources were stained by immunohistochemistry for MHC I (HLA-A,B,C), MHC II (HLA-DP,DQ,DR), and PD-L1 and TC expression was assessed by manual pathologist review. CD8+ cell density was quantified using Definiens image analysis algorithms for the intraepithelial tumor region. RNA extracted from the samples was analyzed by RNAseq, with data available for 48 NSCLC-AD and 46 NSCLC-SQ specimens. A 25-gene IFN-gamma gene signature (IFNG score) was used to study the association of T-cell inflammation with other biomarkers. Results: Of all NSCLC-AD and NSCLC-SQ specimens, 85% showed either complete (<20% TC) or partial (20–80% TC) loss of TC MHC I, while TC MHC II was increased (>1%; MHCIIhi) in 60% of NSCLC-AD and 18% of NSCLC-SQ. Quantitative analysis of CD8 in the tumor microenvironment (TME) revealed a significantly reduced intraepithelial density of CD8+ tumor-infiltrating lymphocytes (TILs) in NSCLC-AD (P=0.009) and NSCLC-SQ (P=0.01) with complete MHC I loss. In contrast, intraepithelial CD8+ TILs were significantly increased in MHCIIhi tumors for both NSCLC-AD (P=0.004) and NSCLC-SQ (P=0.006). Tumors displaying both MHCIIhi and retained MHC I demonstrated the highest TIL density. Gene expression associated with IFN-gamma response was increased in tumors with retained MHC I (P=0.0036) and MHCIIhi samples (P<0.001). In NSCLC-AD and NSCLC-SQ, 38% and 59% had PD-L1 expression ≥1%, respectively. PD-L1 expression correlated with CD8 density and IFNG score in NSCLC-AD, but not in NSCLC-SQ. Conclusions: Understanding the role of the antigen presentation machinery in immune activation and evasion in the TME is important to predict responses to immunotherapy. These data suggest that the expression of MHC I, MHC II, and PD-L1 by tumor cells may play a role in guiding the localization of CD8+ T cells in the TME with differential effects dependent on histologic subtype. The lack of correlation between inflammation and PD-L1 expression in stage I–III NSCLC-SQ suggests an alternate PD-L1 induction mechanism in a subset of those tumors. Citation Format: Cyrus Hedvat, Keyur Desai, Dimple Pandya, Peter Szabo, Johannes Zimmermann, Jan Lesniak, Scott Ely, Sujaya Srinivasan, Xi-Tao Wang, Michele French, Robin Edwards. Tumor intrinsic properties associate with differential effects on CD8+ tumor-infiltrating lymphocyte density and immune gene expression in non-small cell lung cancer (NSCLC) samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1024.

Abstract 5697: Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells

Abstract Background: There is a need for noninvasive predictive biomarkers of response to anti-PD1/PDL1 therapies. Assessment of circulating tumor cells (CTCs) is a rational approach to noninvasive sampling of tumors to understand the potential response or nonresponse to anti-PD1/PD-L1 therapies. IFN-gamma signals through the JAK/STAT cascade to induce PD-L1 via the Interferon Regulatory Factor-1 (IRF1) transcription factor, and is a potent inducer of PD-L1 expression in tumor cells. Recent studies have shown that low or absent IRF1 expression can identify melanomas that lack IFN-gamma responsiveness, and that IRF1 can have a higher predictive value of response to anti-PD1/PD-L1 therapy than PD-L1 itself. Using CTC models and the RareCyte platform, we developed a multiparameter assay that allows simultaneous PD-L1 and IRF1 assessment after CTC identification. We used this assay to better understand the relationship between IRF1 and PD-L1 expression in CTC model systems and applied this assay to blood samples from breast cancer patients. Materials and Methods: Peripheral blood from normal donors or cancer patients under an IRB-approved protocol was collected into RareCyte blood collection tubes. PD-L1(+) and PD-L1(-) CTC models were created by culturing A549 overnight with or without 10ng/mL INF-gamma. The A549 cells were spiked into normal donor blood and buffy coats isolated from 7.5mL of blood by AccuCyte® separation and spread onto slides. MDA-MB-231 cells were used as a model to represent breast cancer cells that express PD-L1 in the absence of IFN-gamma signaling. Slides were stained with a 6-marker panel that included antibodies to pan-cytokeratin (CK), EpCAM, CD45, PDL1, IRF1, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and CTCs identified by CK and/or EPCAM positivity and negative CD45 staining. Confirmed CTCs were then assessed for expression of PD-L1 and IRF1; cellular compartment was recorded for IRF1 staining. Results: Nuclear expression of IRF1 correlated with PD-L1 expression in IFN-gamma stimulated A549 cells. Unstimulated MDA-MB-231 cells expressed high levels of PD-L1, but this did not correlate with nuclear IRF1 expression. Breast cancer patient samples were identified having CTCs that expressed PD-L1. A PDL1 mean fluorescence intensity (MFI) threshold was set at the upper 95% CI of unstimulated A549 cells to define “high” and “low” PD-L1 expression in the patient samples. The population of high-PD-L1 CTCs had higher nuclear IRF1 MFI than low-PD-L1 CTCs, with mean fold-increase of 2.7 (range from 1.4 to 3.3). Conclusions: Nuclear IRF1 expression correlates with PD-L1 expression in breast cancer CTCs. Using CTCs to evaluate expression of IRF1 and PD-L1 together with assessment of CTC mutational load could be a powerful noninvasive assessment of response to anti-PD1/PD-L1 therapies. Citation Format: Lance U'Ren, Nolan Ericson, Ryan Huston, Elisabeth Mahen, VK Gadi, C. Anthony Blau, Eric Kaldjian. Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5697.

Characterization of T Cells Reactive to Hybrid Insulin Peptides in the Peripheral Blood of in Type 1 Diabetes Patients

We recently established, using the NOD mouse model, that hybrid insulin peptides (HIPs), formed spontaneously in islet beta-cells by fusion of insulin C-peptide fragments to peptides of Chromogranin A (ChgA) or Islet Amyloid Polypeptide (IAPP), are ligands for diabetogenic CD4 T cell clones. The goal of this study was to investigate whether HIP-reactive T cells were indicative of ongoing autoimmunity in human T1D patients. To investigate their relevance to human disease, we used IFN-gamma ELISPOT to determine whether HIP-reactive T cells with an inflammatory phenotype were present in PBMCs of new onset T1D patients. We observed that PBMCs from new onset T1D patients responded to 8 of 16 HIPs tested and that nearly half of the patients tested responded to one or more HIPs. We used a CFSE based assay to further characterize T cells reactive to HIP peptides. CD4+CFSEdimCD25+ T cells were sorted single into single wells using FACS and cloned. T cell clonality was confirmed using TCR deep sequencing and T cell reactivity to specific HIPs was confirmed by ELISA. A total of 6 T cell clones were isolated and show a variety of TCR usage. One T cell clone was isolated from the same patient on two different blood draws, indicating a high frequency of this T cell clone in the peripheral blood. While all the T cell clones isolated secreted IFN-gamma in response to HIPs, they also produced other inflammatory cytokines such as TNF-alpha and GM-CSF and reacted to HIPs in the low nanomolar range. Our findings provide new evidence that HIP-reactive T cells are critical players in the pathogenesis of T1D and indicate that HIP-reactive T cells present in the peripheral blood can potentially serve as a biomarker of disease in a subset of patients. Disclosure R.L. Baker: None. T. Delong: None. P. Gottlieb: Advisory Panel; Self; Bristol-Myers Squibb Company. Research Support; Self; Caladrius Biosciences, Inc.. Advisory Panel; Self; Eli Lilly and Company. Board Member; Self; ImmunoMolecular Therapeutics. Consultant; Self; Kamada. Research Support; Self; JDRF, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc., MacroGenics, Inc., Pfizer Inc.. Advisory Panel; Self; Viacyte, Inc.. Research Support; Self; GlaxoSmithKline plc., Janssen Pharmaceuticals, Inc.. K.M. Haskins: None.

Comparative evaluation of immune responses of swine in PRRS-stable and unstable herds

Porcine Reproductive and Respiratory Syndrome (PRRS) is an elusive model of host/virus relationship in which disease is determined by virus pathogenicity, pig breed susceptibility and phenotype, microbial infectious pressure and environmental conditions. Successful disease control in PRRS-endemic Countries corresponds to “stability”, i.e. a condition with no clinical signs of PRRS in the breeding-herd population and no viremia in weaning-age pigs. The aim of this work was to compare the profile and time-course of humoral and cell-mediated immunity in stable and unstable herds, respectively. In particular, we investigated PRRS virus (PRRSV) in serum and group oral fluid samples by Real-time RT-PCR, PRRSV-specific IgA and IgG in oral fluids, serum IgG antibody and the cell-mediated response (PRRSV-specific release of interferon-gamma) in whole blood samples. These parameters were measured in order to identify possible discrepancies in the development and kinetics of the immune response against PRRSV. PRRS-free gilts got regularly infected after entering PRRS-stable and unstable farms. In an open cycle, unstable pig farm PRRSV infection could be demonstrated in all groups of pigs, including suckling piglets. Four main results should be highlighted: A) the precocity of the Ab response in group oral fluids was generally similar to that recorded in sera; B) circulation of PRRSV was consistently detected in all age groups in the unstable herds, as opposed to the stable ones; C) an early, balanced, IgA and IgG response in oral fluids was only observed in the stable herds; D) an early IFN-gamma response after PRRSV infection was often observed in stable herds, as opposed to the unstable ones. These were characterized by IFN-gamma responses in piglets, likely due to transfer of maternal immunity. Most important, the mucosal IgA response was associated with cessation of virus excretion in oral fluid samples of PRRS-unstable herds. The above findings indicate that a peculiar profile of immune response to PRRSV can be found in PRRS-stable herds. Therefore, the outlined immune parameters can represent a useful readout system to evaluate successful adaptation to PRRSV based on acclimatization of breeding animals and management of pig flow.

Dendritic Cell-Based ELISpot Assay for Assessing T-Cell IFN-γ Responses in Human Peripheral Blood Mononuclear Cells to Dengue Envelope Proteins.

Dengue envelope (E) protein is a dominant antigen for vaccine development and E-based vaccines have shown partial or full protection against live-virus challenge in non-human primates. Generally, T cell responses can be investigated with peptides. However, hundreds of over-lapping peptides need to be synthesized to cover the whole sequence of a protein, which brings the cost up to a much higher level than purchasing a protein. We have developed an enzyme-linked immunospot (ELISpot) assay that uses intact E proteins instead of peptides for assessing IFN-gamma (IFN-γ) responses. The assay relies on professional antigen presenting cells, dendritic cells, to process and present the E proteins to stimulate T cells.Peripheral blood mononuclear cells (PBMCs) from dengue-exposed and naïve subjects were selected for the assay development. IFN-γ production ranged from 53 to 513 spot forming units (SFUs) and 0-45 SFUs per million PBMCs in dengue-exposed and naive subject groups, respectively. The assay allowed quantification of E-specific IFN-γ secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Our results suggest that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.

Simulated microgravity ‘disarms’ human Natural Killer cells and suppresses cytotoxic activity against tumor target cells

Maintaining immune system integrity is of paramount importance for astronauts embarking on long-duration spaceflight missions. We have shown that Natural killer (NK) cell function is impaired during spaceflight; however, the mechanisms responsible for this effect are unknown. We exposed primary human blood NK cells to 12 h of simulated microgravity (SMG) using a rotating wall vessel zero gravity cell culture analog. Compared to static and vertical axis rotational controls, SMG (horizontal axis rotation) was found to decrease subsequent NK-cell cytotoxic function in 1G against allogeneic target cell lines of leukemia (K562), multiple myeloma (U266), B-lymphoma (721.221) and HLA-E transfected lymphoma (221.AEH) origin. Flow cytometric analysis revealed that SMG had little effect on the surface expression of a wide-range of NK-cell activating and inhibitory receptors, but did decrease intracellular perforin expression. Using cytometry by time of flight (CyTOF), SMG was found to lower NK-cell degranulation as determined by CD107a expression following co-incubation with K562 target cells in 1G. Moreover, SMG reduced NK-cell expression of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in response to K562 co-culture at 1G. These results indicate that microgravity may be involved in spaceflight-associated reductions in NK cell function. Specifically, pre-exposure to SMG appears to disrupt NK-cell killing by ‘disarming’ them of cytolytic granules and impairing their ability to secrete effector cytokines when confronted with tumor target cells in the 1G environment.

Sense of coherence in lung cancer patients: Association with coping, immune function, and fatigue

Sense of coherence (SOC) reflects the degree to which an individual perceives their world as comprehensible, manageable, and meaningful. Among cancer populations, SOC has been linked with greater quality of life and reduced mortality, suggesting that SOC may serve as a marker of resilience in the context of chronic disease. Utilizing a published adaptation of the transactional model of stress and coping, we hypothesized greater SOC would be associated with adaptive coping, more regulated cytokine response, and lower fatigue. Sixty-two lung cancer patients provided self-report data on SOC, coping, and fatigue. PBMC were incubated with stimulators of acquired and innate immunity (PHA, LPS), and cytokines were measured (pro-inflammatory: IL-1beta IL-6, TNF-alpha; Type 1/TH1: IFN-gamma; Type 2/TH2: IL-5). Linear regressions tested hypotheses. Greater SOC was associated with emotionally expressive coping (p = 0.005), an attenuated IL-5 response to PHA (p = 0.038) and IFN-gamma response to LPS (p=.001); and lower fatigue (p

Abstract 3637: Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules

Abstract Introduction: Immunotherapy with the combination of monoclonal antibodies (mAbs) that block PD-1 (nivolumab) and CTLA-4 (ipilimumab) has shown clinical benefit beyond that observed with either mAb alone. We have developed PD-1 x CTLA-4 bispecific proteins aimed at inducing antitumor immunity through simultaneous blockade of both checkpoint molecules. Two proteins, a tetravalent PD-1 x CTLA-4 bispecific DART® protein (bivalent for both PD-1 and CTLA-4) and a trivalent PD-1 x CTLA-4 TRIDENT™ protein (bivalent for PD-1 but monovalent for CTLA-4) were engineered; the TRIDENT protein was designed to promote CTLA-4 blockade through avidity when bound to PD-1 on cells co-expressing both receptors, thus favoring PD-1 over CTLA-4 blockade in cells that do not co-express both molecules. Methods: Proteins were engineered from humanized PD-1 and CTLA-4 mAb sequences and demonstrated favorable biophysical properties. Binding assays were performed by ELISA or flow cytometry. Co-ligation of PD-1 and CTLA-4 was assessed in an enzyme-fragment complementation assay (DiscoverX). T-cell activation was tested in reporter cells, staphylococcus enterotoxin B-stimulated PBMCs or MLR assays. Results: Both DART and TRIDENT proteins showed equivalent potency in binding immobilized recombinant PD-1 or PD-1-expressing cell lines, inhibition of PD-1 interaction with PD-L1 or PD-L2 as well as reversal of PD-1/PD-L1 mediated T-cell signal inhibition. In all assays, both formats showed activities that were comparable to those of the precursor PD-1 mAb. With respect to CTLA-4, the DART protein showed a minor potency loss in binding to CTLA-4 expressing cells, inhibition of CTLA-4/B7 interaction and reversal of T-cell signal inhibition compared to the precursor mAb. The TRIDENT protein, however, showed substantial lower potency than the DART protein in all CTLA-4 assays, consistent with the monovalent nature of the CTLA-4 arm. Importantly, in cells that co-express both receptors, DART and TRIDENT proteins show comparable co-engagement of PD-1 and CTLA-4, as shown by enzyme-fragment complementation, suggesting that anchoring through PD-1 compensates for the decreased CTLA-4 avidity of the TRIDENT molecule when both target receptors are expressed. Similarly, in T-cell co-activation assays, both DART and TRIDENT proteins showed a comparable increase in IFN-gamma response that recapitulated that observed with the combination of the individual parental mAbs. Conclusion: Both PD-1 x CTLA-4 DART and TRIDENT bispecific molecules block PD-1 and CTLA-4 pathways, with the TRIDENT protein demonstrating a PD-1-biased binding preference, consistent with its design intended to reduce CTLA-4 blockade in the absence of PD-1 co-expression. Both molecules showed comparable T-cell activation activity. Further development of bispecific PD-1 x CTLA-4 molecules for cancer treatment is warranted. Citation Format: Gurunadh R. Chichili, Kalpana Shah, Ross La Motte-Mohs, Alex Berezhnoy, Daorong Liu, Jessica Hill, Leilei He, Christine Shoemaker, Jennifer Brown, Kurt Stahl, Hua Li, Jonathan Li, Sergey Gorlatov, Jim Tamura, Valentina Ciccarone, Ralph Alderson, Ezio Bonvini, Paul A. Moore, Syd Johnson. Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2017-3637

MP48-20 EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TIL) FROM PRIMARY BLADDER TUMORS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2017MP48-20 EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES (TIL) FROM PRIMARY BLADDER TUMORS Michael Poch, Maclean Hall, Krithika Kodumudi Kodumudi, Cortlin Croft, Mayer Fishman, John Mullinax, Amod Sarniak, James Mule, and Shari Pilon-Thomas Michael PochMichael Poch More articles by this author , Maclean HallMaclean Hall More articles by this author , Krithika Kodumudi KodumudiKrithika Kodumudi Kodumudi More articles by this author , Cortlin CroftCortlin Croft More articles by this author , Mayer FishmanMayer Fishman More articles by this author , John MullinaxJohn Mullinax More articles by this author , Amod SarniakAmod Sarniak More articles by this author , James MuleJames Mule More articles by this author , and Shari Pilon-ThomasShari Pilon-Thomas More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1501AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Patients with advanced bladder cancer have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. At our institution, adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has resulted in a durable median OS of 52 months in patients with metastatic melanoma. Immune-mediated anti-tumor responses have been previously shown in bladder cancer, therefore we investigated the phenotype and function of TIL expanded from bladder tumors to establish feasibility of ACT for the treatment of bladder cancer. METHODS Tumor specimens, including primary bladder tumors and lymph node metastases, were collected from 29 bladder cancer patients having standard of care tumor resection, who also had consented to an IRB-approved protocol for TIL generation. The tissue was minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. TIL were considered expanded if they propagated to fill =2 wells. The remaining tumor material was digested into a single cell suspension and frozen. TIL were phenotyped by flow cytometry and assessed for autologous tumor reactivity through co-culture with tumor digest and IFN-gamma ELISA. RESULTS Twenty-seven of 29 tumors were harvested from radical cystectomy patients. Urothelial cell bladder tumors were cultured from 23 patients, of whom 19 (83%) demonstrated TIL expansion. Among these were 9/12 with preceding chemotherapy and 10/11 which had been chemotherapy naive. Expanded TIL were predominantly CD3+ (median 63%, range 10-87%) with a median of 30% CD8+ T cells (range 5-70%). Eight of 15 tested samples (53%) contained TIL that secreted IFN-gamma in response to autologous tumor. Microbial contamination was observed in six specimens. All lymphatic (5/5 samples) tissue collected demonstrated TIL expansion. CONCLUSIONS The study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer. Human bladder cancer tissue can be used to expand tumor-specific TIL in vitro. TIL were also able to be expanded from patients that received chemotherapy prior to tumor resection. Future efforts will explore the ability to further expand bladder TIL cultures to clinically meaningful numbers to develop novel ACT strategies for patients with this diagnosis. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e645 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Michael Poch More articles by this author Maclean Hall More articles by this author Krithika Kodumudi Kodumudi More articles by this author Cortlin Croft More articles by this author Mayer Fishman More articles by this author John Mullinax More articles by this author Amod Sarniak More articles by this author James Mule More articles by this author Shari Pilon-Thomas More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expansion of tumor infiltrating lymphocytes (TIL) from bladder cancer.

142 Background: Patients with advanced bladder cancer have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. At our institution, adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has resulted in a durable median OS of 52 months in patients with metastatic melanoma. Immune-mediated anti-tumor responses have been previously shown in bladder cancer, therefore we investigated the phenotype and function of TIL expanded from bladder tumors to establish feasibility of ACT for the treatment of bladder cancer. Methods: Tumor specimens, including primary bladder tumors and lymph node metastases, were collected from 29 bladder cancer patients having standard of care tumor resection, who also had consented to an IRB-approved protocol for TIL generation. The tissue was minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. TIL were considered expanded if they propagated to fill ≥2 wells. The remaining tumor material was digested into a single cell suspension and frozen. TIL were phenotyped by flow cytometry and assessed for autologous tumor reactivity through co-culture with tumor digest and IFN-gamma ELISA. Results: Transitional cell bladder tumors were cultured from 23 patients, of whom 19 (83%) demonstrated TIL expansion. Microbial contamination precluded TIL growth in six specimens. TIL were cultured from 9/12 (75%) patients with preceding chemotherapy and 10/11 (91%) who were chemotherapy naive. Expanded TIL were predominantly CD3+(median 63%, range 10-87%) with a median of 30% CD8+ T cells (range 5-70%). Eight of 15 tested samples (53%) contained TIL that secreted IFN-gamma in response to autologous tumor. Conclusions: The study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer. Human bladder cancer tissue can be used to expand tumor-specific TIL in vitro. TIL were also expanded from patients that received chemotherapy prior to tumor resection. Future efforts will explore the ability to further expand bladder TIL cultures to clinically meaningful numbers to develop novel ACT strategies for patients with this diagnosis.

Assessment of Cytokine mRNA Expression Profiles in Tumor Microenvironment and Peripheral Blood Mononuclear Cells of Patients with High-grade Serous Carcinoma of the Ovary

Objective: Tumor establishment, metastatic spreading and poor survival in ovarian cancer is strongly associated with progressive derangement of the patient’s immune system. Accumulating evidence suggests that immune impairment is influenced by the production and presence of cytokines in the tumor microenvironment. Methods: Cytokine mRNA profiles in tumor tissue and peripheral blood mononuclear cells (PBMC) were analyzed in patients with high grade serous carcinoma (HGSC) of the ovary and compared it to patients with benign ovarian conditions and controls with normal ovaries. Cytokine assessment was done by real-time quantitative RT-PCR and specific primers and probes for 12 cytokines-IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β/LTA, TGF-β1, and GM-CSF chosen to distinguish between cytotoxic Th1, humoral Th2, regulatory Th3/Tr1 and inflammatory responses. Results: The cytokine mRNA response in the HGSC patients was significantly up regulated compared to patients with benign ovarian conditions and normal ovary controls confirming the immunogenicity of HGSC and implying immune recognition and reaction locally in the tumor microenvironment and systemically in the peripheral blood.There was an up-regulation of inflammatory and inhibitory cytokine mRNA promoting tumor progression, T-regulatory cell priming and T-regulatory cell-mediated immune suppression. In contrast, there was an inability to mount the crucially important IFN gamma response needed for upregulation of the cytotoxic anti-tumor response in the local microenvironment. In addition, systemic IL-4- mediated Th2 response prevailed in the peripheral blood deviating the systemic defense towards humoral immunity. Conclusions: Taken together, these results suggest local and systemic cytokine cooperation promoting tumor survival, progression and immune escape. Our study confirms and extends previous investigations and contributes to the evaluation of potential cytokine candidates for diagnostic cytokine mRNA profiles and for future therapeutic interventions based on cytokine inhibition.

Gene electrotransfer into skin using noninvasive multi-electrode array for vaccination and wound healing

Skin is an attractive target for gene electrotransfer due to its easy accessibility and its interesting immune properties. Since electrodes are often invasive and frequently induce discomfort during pulse application, there is a fundamental need for non-invasive electrodes for skin delivery. We developed circular pin non-invasive multi-electrode array (MEA), suitable for different clinical applications. MEA was first employed to deliver a luciferase reporter gene. Then, it was used to deliver a DNA vaccine coding for ovalbumin or a plasmid encoding hCAP-18/LL-37 for promoting wound healing. The results demonstrated a strong gene expression and an efficient delivery of both, DNA vaccine and wound healing agent, dependent on the pulses applied. The use of MEA to deliver the ovalbumin plasmid demonstrated a strong immune response, as evidenced by the presence of antibodies in sera, the IFN-gamma response and the delayed tumor growth when the mice were subsequently challenged with B16-OVA cells. The delivery of a plasmid encoding hCAP-18/LL-37 significantly accelerated wound closure. The easy applicability and non-invasiveness of MEA make it suitable for various clinical applications that require gene electrotransfer to skin. Specifically, by adapting electric pulses to the expected action of a transgene, non-invasive MEA can be employed either for vaccination or for wound healing.

Distinct functional enrichment of transcriptional signatures in pigs with high and low IFN-gamma responses after vaccination with a porcine reproductive and respiratory syndrome virus (PRRSV).

Little is known about the host factor in the response to PRRSV vaccination. For this purpose, piglets were immunized with a commercial PRRSV-live vaccine and classified as high responders (HR) or low responders (LR) as regards to the frequencies of virus-specific IFN-γ-secreting cells. Six weeks post vaccination, PBMCs isolated from three individuals with the most extreme responses in each HR and LR groups and 3 unvaccinated controls, were either stimulated with phytohaemagglutinin, challenged with the vaccine or mock treated for 24 h, prior conducting transcriptional studies, gene ontology and pathway analyses. The LR group had very low neutralizing antibody levels and showed a higher number of down-regulated transcripts compared with the HR group (FDR < 0.2, P < 0.001). Down-regulated genes encoded chemoattractants, proinflammatory cytokines and the interferon-inducible GBP family, and showed enrichment in wounding (FDR < 3.6E-13), inflammation (FDR < 8E-12), defence (FDR < 8.7E-09) and immunity (FDR < 7.6E-08), suggesting immune response impairment. In the HR group, down-regulated genes were involved in protein transport (FDR < 4.77E-03), locomotory behavior (FDR < 5.47E-3), regulation of protein localization (FDR < 1.02E-02), and regulation of TNF superfamily member 15 and miR181. In contrast, the HR group presented up-regulated transcripts associated with wounding (FDR < 4.95). Moreover, IFN-γ was predicted to be an inhibited upstream regulator since IFN-γ pathways were associated with higher number of down-regulated genes in the LR (n = 40) than the HR (n = 10). Divergent responses to PRRSV-vaccination may be the result of the genetic background of the host.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0392-3) contains supplementary material, which is available to authorized users.

Exosomes in human semen impair antigen-presenting cell function and decrease antigen-specific T cell responses

Abstract Exposure to semen is the primary route of transmission for many sexually transmitted infections. Accumulating evidence suggests that components in semen directly impair leukocytes, which could compromise the protective efficacy of vaccine-induced immune responses in the mucosa. Exosomes, small microvesicles with immunomodulatory functions, are present at an average concentration of 2.2 × 1013 particles per ejaculate (n = 18). These seminal exosomes (SE) efficiently and rapidly entered peripheral and vaginal dendritic cells (DCs), whereas T cell uptake was poor. In PBMC cultures, SE impaired memory T cell function, reducing the production of TNF-alpha, IL-2 and/or IFN-gamma in response to virus-derived peptides an average of 60% for CD4+ T cells and 48% for CD8+ T cells, in a dose-responsive manner (n = 6). Reduction of cytokine production by memory CD8+ T cells was reduced to a greater extent for protein antigens as compared to peptide antigens, implying that antigen-presenting cell function was impaired by the presence of SE. Exposing only DCs, as opposed to bulk PBMCs, to SE also blocked subsequent CD8+ memory T cell activation, reducing the proportion of cells making cytokines by 22% (n = 5 donors). Expression of CD107a, a marker of cytotoxic T cell degranulation, was decreased by 42% in PBMC cultures exposed to SE and superantigen (n=4). SE also impaired vaginal T cell cytokine and degranulation responses to superantigen. Thus, SE likely inhibit antigen-specific T cell responses by impairing antigen-presenting cell function. Understanding how programmed immune responses are altered by the presence of semen is important to developing the next generation of vaccine and preventative treatments against sexually transmitted disease

Two-Year Follow-up Study of Mycobacterium tuberculosis Antigen-Driven IFN-γ Responses and Macrophage sCD14 Levels After Tuberculosis Contact.

Clinical data regarding the prediction of active tuberculosis (TB) development in close TB contacts are scarce. To address this problem, we performed a 2-year follow-up study of Mycobacterium tuberculosis (M.tb) antigen-driven IFN-gamma responses and serum levels of soluble macrophage CD14 receptor in individuals with recent or prolonged M.tb exposure. Between June 2011 and June 2013, we studied 60 healthy Polish adults with recent household or long-term work TB contact and individuals without known M.tb exposure. All of them underwent baseline and repeated testing with IGRA (IFN-gamma release assay) and serum sCD14 ELISA quantification. Frequencies of IGRA results differed at the baseline and follow-up testing. IGRA reversions were noticed in almost one-third of Work TB Contacts and no participants from the Household TB Contact group. IGRA conversions were found in 40% of Household TB Contacts. No correlation between the IGRA result and the sCD14 level was observed. IFN-γ variability has important implications for clinical practice and requires caution in interpreting the results to distinguish new infections from nonspecific inter-individual variations in cytokine responses. The impairment of IFN-γ response in some individuals with prolonged M.tb exposure representing a resistant immune status does not allow considering IGRA results as reliable and credible. Monitoring the serum sCD14 level can reduce the likelihood of a false prediction of active TB development in close TB contacts showing an M.tb-specific increase in the IFN-gamma production in repeated IGRA testing.

JQ1, a Selective Bromodomain Inhibitor, Augment the Immunogenicity of Mantle Cell Lymphoma By Influencing the Expression of PD-L1

Mantle cell lymphoma (MCL) is a rare and aggressive form of non-Hodgkin's B-cell lymphoma and represents 4% to 8% of NHL. New therapeutic strategies are under development to improve the clinical outcome of patients with this disease. Recent studies have highlighted the importance of BRD/acetyl-lysine binding in orchestrating molecular interactions in chromatin biology and regulating gene transcription. The Bromodomain (BRD) is a diverse family of evolutionary conserved protein-interaction modules, which recognizes acetylated lysine residues such as those on the N-terminal tails of histones.We have recently determined the functional consequences of inhibiting Bromodomains in MCL. First, we evaluated the effects of JQ1, a selective small-molecule bromodomain inhibitor upon MCL's immunogenicity and antigen-specific CD4+ T-cell responses. In vitro treatment of MCL cells with increasing concentrations of JQ1 resulted in a decreased expression of the tolerogenic molecule PD-L1 as compared to control cells. Second, we evaluated the capabilities of JQ1-treated MCL cells to present cognate antigen to naïve or anergic antigen-specific CD4+ T-cells. We found that treatment of MCL cells with JQ1 enhances their antigen-presenting capabilities leading to effective priming of naïve CD4+ T-cells and restoring the responsiveness of tolerant T-cells as determined by their increased production of IL-2 and IFN-gamma in response to cognate antigen. More importantly, in vivo studies clearly demonstrated a strong anti-tumor effect in JQ1-treated MCL tumor bearing mice. Mechanistically, we found a decreased STAT3 phosphorylation and c-Myc expression in JQ1-treated MCL cells, which in turn might be responsible for the diminished expression of PDL1 and augmentation of MCL's immunogenicity.Taken together, we have found that MCL cells treated with the Bromodomain specific inhibitor JQ1 are more inflammatory, display lower expression of the immunosuppressive molecule PDL1 and, are capable of restoring the responsiveness of tolerant T-cells. More importantly, JQ1 treatment in vivo inhibits tumor growth in MCL tumor bearing animals. Our studies therefore have unveiled a previously unknown immunological effect of BRD inhibitors and have identified a novel immunotherapeutic approach in MCL. DisclosuresNo relevant conflicts of interest to declare.

Association of CMV-Specific T Cell-Mediated Immunity with CMV DNAemia and Development of CMV Disease in HIV-1–Infected Individuals

BackgroundAmong HIV-1–infected individuals, cytomegalovirus (CMV) reactivation and disease occur in the setting of advanced immunosuppression. The value of a standardized assessment of CMV-specific T-cell mediated immunity by the CMV QuantiFERON assay (CMV-QFT) has not yet been thoroughly investigated in HIV-1–infected subjects.MethodsProspective, longitudinal study in 153 HIV-1–infected subjects with a CD4+ T cell count < 350/μL who simultaneously underwent CMV-QFT, CMV serology testing and CMV-DNA quantification. Factors associated with CMV-QFT were evaluated. Clinical screening for CMV manifestations was then performed every 3 months.ResultsAmong the 141 CMV IgG-seropositive individuals the CMV-QFT assay yielded reactive results in 84% (118/141), negative results in 15% (21/141) and indeterminate (negative mitogen IFN-gamma response) results in 1% (2/141) of subjects. The mean actual CD4+ T cell count was significantly higher in CMV-QFT reactive subjects, when compared to CMV-QFT non-reactive individuals (183 ± 102 vs. 126 ± 104 cells/μL, P = 0.015). A significantly lower proportion of CMV-QFT reactive vs. non-reactive patients displayed CMV DNAemia > 100 copies/mL (23% (27/118) vs. 48% (11/23), P = 0.02). Furthermore, a statistically significant inverse association between mitogen IFN-gamma response and CMV-DNAemia > 1000 copies/mL was observed (P < 0.001). During the observational period, 5 CMV end-organ manifestations were observed. In three of the CMV cases the CMV-QFT yielded indeterminate results.ConclusionsWhile CMV-QFT reactivity indicates CMV-specific immunity, indeterminate results due to negative mitogen IFN-gamma response might reflect HIV-1-induced immunodeficiency. Thus, dependency upon CD4+ T cell count should be considered when interpreting CMV-QFT results.

Abstract 3136: Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma

Abstract Bone marrow (BM) serves as a reservoir for a unique population of memory T cells. Tumor-specific T cells have been identified in the bone marrow of cancer patients. However, few direct functional analyses of tumor-specific responses in T cells isolated from BM have been performed. In this study, we investigated the ability of bone marrow T cells to induce anti-tumor immunity in mice bearing neuro2A neuroblastoma or B16 melanoma. To demonstrate the presence of antigen-specific T cells in BM, we first examined the percent of antigen-specific CD8+ T cells in vaccinated mice. The overall percent of antigen-specific CD8+ T cells was much higher in BM compared to spleen and lymph nodes of vaccinated mice. To investigate the ability of antigen-specific BM T cells to induce cytotoxic function, BM T cells were isolated from mice vaccinated with OVASIINFEKL peptide pulsed dendritic cells (DC). Expanded BM T cells from DC vaccinated mice co-cultured with M05 or irrelevant tumor cells led to tumor-specific cytotoxicity (p&amp;lt;0.001). In B16 tumor-bearing mice, T cells isolated from BM produced IFN-gamma in response to gp100 peptide (1400+210 pg/ml). Adoptive transfer of BM T cells isolated from B16-bearing mice led to a delay in tumor growth compared to control groups (p&amp;lt;0.001). In the neuroblastoma model, T cells isolated from BM of neuro2A-bearing mice produced higher levels of IFN-gamma and were tumor-specific (250+90 pg/ml, p&amp;lt;0.01 compared to T cells cultured with irrelevant tumor cells). Adoptive transfer of expanded BM T cells and DC vaccination in the setting of BM transplant led to a significant delay in tumor growth and enhanced survival in neuro2A bearing mice compared to BM transplant + DC vaccination alone group (p&amp;lt;0.02). These studies demonstrate that tumor-specific T cells can be isolated from the BM of tumor-bearing mice. Adoptive transfer of T cells expanded from the BM can delay tumor growth and improve survival. These studies support the isolation and expansion of T cells from the BM for use in adoptive cell therapy strategies in patients with neuroblastoma and melanoma. Citation Format: Krithika N. Kodumudi, Amy Weber, Ellen Moore, Amod Sarnaik, Shari Pilon-Thomas. Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3136. doi:10.1158/1538-7445.AM2015-3136