Costimulatory Molecule 4-1BBL Research Articles

Highly-Multiplexed Immunofluorescence PhenoCycler Panel for Murine FFPE Yields Insight into Tumor Microenvironment Immunoengineering

Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is Co-Detection-by-indEXing (CODEX), recently renamed as the PhenoCycler system. This is a highly-multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh-frozen tissues. Formalin-fixed paraffin-embedded (FFPE) blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine FFPE tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of three gene-delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift towards intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of IFNγ, in addition to 4-1BBL and IL-12, further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help to explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of IFNγ therapeutic efficacy.

The role of IL‑17, IFN‑γ, 4‑1BBL and tumour‑infiltrating lymphocytes in the occurrence, development and prognosis of pancreatic cancer.

Immunotherapy has made progress in the treatment of tumours; however, in patients with pancreatic cancer, immunotherapy has not achieved effective results. The present study investigated changes in the immune microenvironment during tumour development and progression, and the relationship between the immune microenvironment and prognosis, to clarify the mechanism of immune escape in pancreatic cancer. A total of 40 patients with pancreatic cancer (including 22 with stage I-II disease and 18 with stage III-IV disease) and 20 patients with chronic pancreatitis were included in the present study. The expression of CD3, CD4, CD8, CD56, IFN-γ, IL-17 and 4-1BBL was assessed by immunohistochemistry, and the mRNA expression levels were detected by reverse transcription-quantitative PCR (RT-qPCR). The clinicopathological characteristics and prognoses of patients with pancreatic cancer were analysed to further explore the role of IL-17, IFN-γ, 4-1BBL and tumour-infiltrating lymphocytes in pancreatic cancer. Notably, the expression levels of CD3, CD8, CD56, IFN-γ and 4-1BBL in patients with stages I-II and III-IV cancer were lower than those in patients with chronic pancreatitis (P<0.05), especially in patients with stage III-IV cancer (P<0.05). In addition, the expression of IL-17 in patients with stages I-II and III-IV cancer was greater than in patients with chronic pancreatitis (P<0.05), especially in patients with stage III-IV cancer (P<0.05). The RT-qPCR results regarding CD3, CD4, CD8, CD56, IFN-γ and IL-17 were almost the same as those obtained from immunohistochemical analysis; however, the mRNA expression levels of 4-1BBL were not significantly different between stages I-II and III-IV. Furthermore, patients with pancreatic cancer with higher expression levels of CD3, CD8, CD56, IFN-γ and 4-1BBL exhibited longer survival, whereas those with higher expression of IL-17 had a shorter survival time. The expression levels of CD3, CD8, CD56, cytokines IL-17 and IFN-γ, and costimulatory molecule 4-1BBL were revealed to be related to the degree of differentiation, Tumour-Node-Metastasis staging and the prognosis of pancreatic cancer, and may serve as novel immunological indicators for evaluating the condition and treatment effectiveness in patients with pancreatic cancer.

Polymeric nanoparticle gel for intracellular mRNA delivery and immunological reprogramming of tumors

Immuno-oncology therapies have been of great interest with the goal of inducing sustained tumor regression, but clinical results have demonstrated the need for improved and widely applicable methods. An antigen-free method of cancer immunotherapy can stimulate the immune system to recruit lymphocytes and produce immunostimulatory factors without prior knowledge of neoantigens, while local delivery reduces the risk of systemic toxicity. To improve the interactions between tumor cells and cytotoxic lymphocytes, a gene delivery nanoparticle platform was engineered to reprogram the tumor microenvironment (TME) in situ to be more immunostimulatory by inducing tumor-associated antigen-presenting cells (tAPCs) to activate cytotoxic lymphocytes against the tumor. Biodegradable, lipophilic poly (beta-amino ester) (PBAE) nanoparticles were synthesized and used to co-deliver mRNA constructs encoding a signal 2 co-stimulatory molecule (4-1BBL) and a signal 3 immuno-stimulatory cytokine (IL-12), along with a nucleic acid-based immunomodulatory adjuvant. Nanoparticles are combined with a thermoresponsive block copolymer for gelation at the injection site for local NP retention at the tumor. The reprogramming nanoparticle gel synergizes with immune checkpoint blockade (ICB) to induce tumor regression and clearance in addition to resistance to tumor rechallenge at a distant site. In vitro and in vivo studies reveal increases in immunostimulatory cytokine production and recruitment of immune cells as a result of the nanoparticles. Intratumoral injection of nanoparticles encapsulating mRNA encoding immunostimulatory agents and adjuvants via an injectable thermoresponsive gel has great translational potential as an immuno-oncology therapy that can be accessible to a wide range of patients.

Abstract LB223: Intratumoral administration of modified vaccinia Ankara expressing a tumor associated antigen and the costimulatory molecules IL-12 and 4-1BBL induces potent systemic long-term immune responses against murine tumors

Abstract In the last decade, immunotherapy has revolutionized cancer treatment. However, the lack of potent therapy-induced immune responses against solid tumors due to the immune-suppressive tumor microenvironment (TME) is still a major drawback. One approach to reprogram TME is virotherapy, which can target tumors via destruction of infected tumor cells as well as activating immune cell populations in the TME. A versatile and safe tool for virotherapy is modified vaccinia virus Ankara (MVA), which has already been approved by the FDA as a non-replicating vaccine against smallpox and monkeypox and is also in clinical trials against other infectious diseases and cancer. In the current study, we deciphered the anti-tumorigenic effect of a novel MVA vector encoding a tumor associated antigen (TAA) and the costimulatory molecules IL-12 and 4-1BBL, called MVA-TAA-4-1BBL-IL12. We could show that repetitive intratumoral (IT) injection of MVA-TAA-4-1BBL-IL12 resulted in strong tumor growth control, shrinkage and/or complete elimination of tumors and induction of tumor-specific CD8 T cell response. Furthermore, MVA-TAA-4-1BBL-IL12 treatment promoted a systemic anti-tumor immune response not only against the treated tumor but also untreated, distant tumors. Importantly, local virotherapy also resulted in the generation of tumor-specific memory response, which protected mice against local recurrence after rechallenge. As a conclusion, our findings show that MVA-TAA-4-1BBL-IL12 IT treatment induces an effective anti-tumorigenic immune response in different solid tumor models, which gives rise to long-term protection against cancer recurrence. Citation Format: Cigdem Atay Langbein, Raphael Giessel, Mahmad Ismail Housni, Kerstin Lämmermann, Barbara Bathke, André Riedl, Jürgen Hausmann, Markus Kalla, Paul Chaplin, Hubertus Hochrein, José Medina-Echeverz, Maria Hinterberger. Intratumoral administration of modified vaccinia Ankara expressing a tumor associated antigen and the costimulatory molecules IL-12 and 4-1BBL induces potent systemic long-term immune responses against murine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB223.

Dysfunctional Sars-CoV-2-M protein-specific cytotoxic T lymphocytes in patients recovering from severe COVID-19

Although the importance of virus-specific cytotoxic T lymphocytes (CTL) in virus clearance is evident in COVID-19, the characteristics of virus-specific CTLs related to disease severity have not been fully explored. Here we show that the phenotype of virus-specific CTLs against immunoprevalent epitopes in COVID-19 convalescents might differ according to the course of the disease. We establish a cellular screening method that uses artificial antigen presenting cells, expressing HLA-A*24:02, the costimulatory molecule 4-1BBL, SARS-CoV-2 structural proteins S, M, and N and non-structural proteins ORF3a and nsp6/ORF1a. The screen implicates SARS-CoV-2 M protein as a frequent target of IFNγ secreting CD8+ T cells, and identifies M198–206 as an immunoprevalent epitope in our cohort of HLA-A*24:02 positive convalescent COVID-19 patients recovering from mild, moderate and severe disease. Further exploration of M198–206-specific CD8+ T cells with single cell RNA sequencing reveals public TCRs in virus-specific CD8+ T cells, and shows an exhausted phenotype with less differentiated status in cells from the severe group compared to cells from the moderate group. In summary, this study describes a method to identify T cell epitopes, indicate that dysfunction of virus-specific CTLs might be an important determinant of clinical outcomes.

Ovarian Cancer Immunotherapy en Route: IL9 Inhibits Growth of Ovarian Cancer and Upregulates its Expression of Ox40L and 4-1BBL

Objective: Ovarian cancer is the deadliest of all gynecologic tumors. Current treatment methods include debulking surgery with chemotherapy, however even with treatment, the five-year survival rate is below 45%. Cancer immunotherapy is an innovative treatment option being highly researched. Interleukins (ILs) are signaling molecules used by the human immune system to assist in detecting and destroying cancer cells. The ability of tumor cells to evade the immune system is a major challenge we face in fighting cancer. Ox40L/Ox40 and 4-1BBL/4-1BB are key immune costimulatory molecules that increase T cell activation to eliminate tumors. Past research has shown that IL9 has unique influences on various types of cancer, however, its role in ovarian cancer has not yet been assessed. In this study, ovarian cancer cells were treated with IL9 and the expression of Ox40L and 4-1BBL were measured. Methods: A2780 ovarian cancer cells were treated with IL9. Proliferation of ovarian cancer cells was measured by a Clonogenic Survival Assay and Quick Proliferation Assay. RT-PCR was conducted to determine whether IL9 upregulated the costimulatory molecules Ox40L and 4-1BBL. IHC was performed to further investigate IL9 upregulation of Ox40L and 4-1BBL. Results: Treatment of A2780 ovarian cancer cells with IL9 resulted in decreased proliferation of the ovarian cancer cells. By using RT-PCR, it was determined that IL9 treated ovarian cancer cells displayed upregulation of the costimulatory molecules Ox40L and 4-1BBL. Upregulation of OX40L and 4-1BBL was further confirmed by IHC. Conclusions: IL9 inhibited growth of ovarian cancer cells, and IL9 upregulated the key immune costimulatory molecules Ox40L and 4-1BBL. This suggests that increased expression of Ox40L and 4-1BBL may be associated with the inhibitory effect of IL9 on proliferation of ovarian cancer. This study warrants further investigation of the role of Ox40L and 4-1BBL in ovarian cancer growth.

Abstract 1689: Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression

Abstract Immunostimulatory gene therapy using oncolytic viruses - OV - for combined gene transfer and tumor-specific oncolysis is a promising immunotherapy for cancer. Due to neutralizing antibodies in the blood, OVs are commonly administered by intratumoral delivery. The immune activation is both local and systemic as activated dendritic cells in the injected lesion travel to lymph nodes to activate tumor-reactive T cells that can in turn act on both injected and non-injected lesions. In the present project, we demonstrate that OV-infected tumor cells released exosomes that cargo mRNA and protein coded by the human immunostimulatory genes expressed by the OV. Tumor cell-derived exosomes are commonly immunosuppressive because they cargo immunosuppressive molecules expressed by the tumor. We hypothesized that exosomes derived from OV-infected tumor cells may instead be immunostimulatory. To investigate the role of exosomes derived from OV-treated tumors, human melanoma Mel526 cells were infected by delolimogene mupadenorepvec -LOAd703 - which is an adenoviral serotype 5/35 OV armed with costimulatory molecules CD40L and 4-1BBL. LOAd703 could induce expression of CD40L and 4-1BBL on the melanoma cell surface and kill the cells by replication-induced oncolysis after 48-72 hours culture as evaluated by MTS. Exosomes were purified prior oncolysis and investigated for the presence of CD40L and 4-1BBL mRNA and protein, and for their capacity to stimulate immune responses. The results demonstrated that the exosomes can indeed cargo CD40L and 4-1BBL both as mRNA as shown by PCR, and protein as shown by ELISA, western blot, flow cytometry and electron microscopy post LOAd703 tumor cell infection. Furthermore, the exosomes from CD40L and 4-1BBL-expressing tumor cells could stimulate robust dendritic cell activation with enhanced levels of MHC class I and II as well as costimulatory molecules CD80, CD86 and CD83. In contrast, MHC class I and the costimulatory molecules were reduced on dendritic cells stimulated with exosomes derived from non-infected tumor cells, which is in line with previous knowledge of exosomes as immunosuppressants in the tumor. However, PD-L1 was increased on dendritic cells stimulated with the exosomes from CD40L and 4-1BBL-expressing tumor cells. Hence, LOAd703 therapy may benefit from combination with PD-L1 or PD-1 checkpoint blockade. In conclusion, tumor-derived exosomes released by OV-infected tumor cells can cargo the immunostimulatory transgenes expressed by the OVs and are then converted from immunosuppressive to immune activating exosomes as shown by their capacity to activate immune cells such as dendritic cells. Exosomes that cargo immunostimulatory transgenes from OV-infected tumor cells may support the systemic immunity gained by OVs after local intratumoral delivery. Citation Format: Alireza Labani-Motlagh, Sedigheh Naseri, Jessica Wenthe, Emma Eriksson, Angelica Loskog. Exosomes derived from tumor cells treated with immunostimulatory oncolytic virotherapy exert immune activation instead of immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1689.

The IL15 Superagonist Complex ALT-803 Potently Enhances the Proliferative Capacity and Activity of in Vitro Expanded Natural Killer Cells

IntroductionWe previously reported on the treatment of 8 relapsing high-risk multiple myeloma (HRMM) patients with ex vivo activated/expanded NK cells (ENKs). We demonstrated that interleukin-2 (IL2) provided suboptimal cytokine support post adoptive transfer of ENKs. Aiming to improve upon prior data we hypothesize that the novel IL15 superagonist complex ALT-803 will provide superior support compared to IL2 and enhance ENK activity, proliferation and persistence.MethodsTo compare the activity of IL2 and ALT-803 on ENKs, we performed long-term in vitro culture studies. Analogous to the 8-day clinical production method, healthy donor (HD) peripheral blood mononuclear cells (PBMCs) were first co-cultured with the leukemic cell line K562 modified to express both membrane-bound IL15 and the costimulatory molecule 4-1BBL, and high-dose IL2. We deliberately chose to maintain IL2 in the expansion method rather than substituting ALT-803 because our FDA-approved GMP-grade expansion was validated using IL2. On expansion Day 8 (the day clinical ENK products are infused), the ENKs were either maintained at high-dose (300U/mL) IL2, or otherwise cultured at low-dose (20U/mL) IL2 or 35ng/mL ALT-803, in all conditions without K562 stimulator cells. Proliferation (fold-change from baseline) was measured on Days 14, 21, and 28, and expression of key activating and inhibitory markers was assessed via flow cytometry. Relative cytolytic ability was determined via 4-hour chromium (Cr)-release assays against K562 and the MM cell lines OPM2, JJN3, and U266.ResultsProliferation of ALT-803-ENKs was superior to both IL2-ENK conditions at all assessed time points; on Day 28, fold-expansion of ALT-803-ENKs was 753, versus 156- and 557-fold for ENKs maintained in 20U or 300U/mL IL2, respectively, Figure 1A. Immunophenotyping studies demonstrated significant differences between ENKs cultured in 20U/mL IL2 versus ALT-803-treated ENKs, including activation (CD26, CD69, DNAM1, NKp30) and functional (CD16, perforin, granzyme, TRAIL) molecules. ALT-803-treated ENKs maintained an activated phenotype, whereas IL2 ENKs (20U/mL) exhibited reduced expression of several markers by Day 14, with further decreases noted on Days 21 and 28. Representative examples are depicted in Figure 1B. Morphologically, IL2-ENKs acquired apoptotic features after extended culture, whereas ALT-803-ENKs did not. Though cytolytic ability was variable on Days 14 and 21, the activity of ALT-803-ENKs exceeded that of IL2-ENKs (20U/mL) against K562, OPM2, JJN3, and U266 cell lines on Day 28. High-dose IL2-ENKs exhibited a similar phenotype and cytolytic ability compared to ALT-803-ENKs.ConclusionsWe compared clinically-relevant doses of IL2 and ALT-803 to determine whether ALT-803 is superior at maintaining ENK activity and proliferative capacity. ALT-803 ENKs exhibited robust proliferation through Day 28 and sustained expression of key activation and functional markers, highlighting the superiority of ALT-803 in this setting. After four weeks of continuous culture, ALT-803-ENKs maintained greater cytolytic ability against MM cell lines. Though high-dose IL2 is capable of sustaining ENK activation state in vitro, this dose is not achievable in humans. Unlike IL2, ALT-803 does not induce expansion of T regulatory cells, and has a more favorable toxicity profile. Importantly, ALT-803 stimulated and maintained expression of CD16, indicating that it would be well-suited to pair with monoclonal antibodies for the support of antibody-dependent cellular cytotoxicity. Further studies are in progress to study the effects of ALT-803 on ENKs in the presence of suppressive cytokines/cells to determine if ALT-803 is able to overcome the effects of the tumor microenvironment. [Display omitted] DisclosuresWong:Altor BioScience: Employment, Equity Ownership. Jeng:Altor Bioscience: Employment, Equity Ownership. Shrestha:Altor Bioscience: Employment, Equity Ownership. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Abstract A06: Direct and indirect effects of low-dose radiation on Tregs and antitumor efficacy among diverse tumor models

Abstract The impact of radiation on TREG cells remains controversial. Radiation has been shown by our lab, and others, to alter the phenotype of human tumor cell by enhancing their expression of the co-stimulatory molecules OX-40L and 4-1BBL. Both OX-40 and 4-1BB signaling have been reported to influence TREG frequency and function, and we hypothesized that radiation-induced changes in expression of these molecules could influence the frequency of TREG cells within tumors. We evaluated three widely used murine tumor models for these experiments. 4T1 (mammary), MC38 (colon), or CT26 (colon) cells were implanted s.c. into mice and treated locally with 8-10Gy of radiation. Several days after treatment the frequency of tumoral T cells, including CD4+Foxp3+ TREG cells, was evaluated by fluorescent IHC and flow cytometry. We found that radiation differentially impacted the frequency of TREGS among the three tumor models evaluated and appeared to correlate with expression levels of 4-1BBL and OX-40L on the tumor cells post-IR. The level of tumor control post-RT also mirrored expression levels of 4-1BBL and OX-40L on tumor cells. Interestingly, 4T1 tumors contained more total CD4+ cells within tumors, but contained less TREG cells within the CD4 population, after radiation as compared to MC38 tumors. 4T1 tumor-bearing mice also contained more CD4+41BB+ cells as compared to MC38 tumor-bearing mice. These differences may contribute to differences in the therapy responses of various murine models utilizing radiation and may serve as useful biomarker to predict antitumor responses post-IR. Citation Format: Samantha Simon, Charlie Garnett-Benson. Direct and indirect effects of low-dose radiation on Tregs and antitumor efficacy among diverse tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A06.

Nuclear-localized costimulatory molecule 4-1BBL promotes colon cancer cell proliferation and migration by regulating nuclear Gsk3β, and is linked to the poor outcomes associated with colon cancer

ABSTRACT Anti-tumor immune response and the prognosis of tumor are the results of competition between stimulatory and inhibitory checkpoints. Except for upregulating inhibitory checkpoints, lowering some immune accelerating molecules to convert an immunostimulatory microenvironment into an immunodormant one through “decelerating the accelerator” might be another effective immune escape pattern. 4-1BBL is a classical transmembrane costimulatory molecule involving in antitumor immune responses. In contrast, we demonstrated that 4-1BBL is predominantly localized in the nuclei of cancer cells in colon cancer specimens and is positively correlated with tumor size, lymph node metastasis, and a lower survival ratio. Furthermore, the nuclear localization of 4-1BBL was also ascertained in vitro. 4-1BBL knockout (KO) arrests the proliferation and impaired the migration and invasion ability of colon cancer cells in vitro and retarded tumor growth in vivo. 4-1BBL KO increased the accumulation of Gsk3β in the nuclei of colon cancer cells and consequently decreased the expression of Wnt pathway target genes and thus alter tumor biological behavior. We hypothesized that unlike membrane-expressed 4-1BBL, which stimulates the 4-1BB signaling of antitumor cytotoxic T cells, the nuclear-localized 4-1BBL could facilitate the malignant behavior of colon cancer cells by circumventing antitumor signaling and driving some key oncotropic signal pathway in the nucleus. Nuclear-localized 4-1BBL might be an indicator of colon cancer malignancy and serve as a promising target of immunotherapy.

A novel Epstein\u2013Barr virus-latent membrane protein-1-specific T-cell receptor for TCR gene therapy

Background:Adoptive transfer of genetically engineered T-cells to express antigen-specific T-cell receptor (TCR) is a feasible and effective therapeutic approach for numerous types of cancers, including Epstein–Barr virus (EBV)-associated malignancies. Here, we describe a TCR gene transfer regimen to rapidly and reliably generate T-cells specific to EBV-encoded latent membrane protein-1 (LMP1), which is a potential target for T-cell-based immunotherapy.Methods:A novel TCR specific to LMP1 (LMP1-TCR) was isolated from HLA-A*0201 transgenic mice that were immunised with the minimal epitope LMP1166 (TLLVDLLWL), and LMP1-TCR-transduced peripheral blood lymphocytes were evaluated for functional specificities.Results:Both human CD8 and CD4 T-cells expressing the LMP1-TCR provoked high levels of cytokine secretion and cytolytic activity towards peptide-pulsed and LMP1-expressing tumour cells. Notably, recognition of these T-cells to peptide-pulsed cells was maintained at low concentration of peptide, implying that the LMP1-TCR has high avidity. Infusion of these engineered T-cells revealed remarkable therapeutic effects and inhibition of tumour growth in a preclinical xenogeneic model. We observed explosive ex vivo proliferation of functional TCR-transduced T-cells with artificial antigen-presenting cells that express co-stimulatory molecules CD80 and 4-1BBL.Conclusions:These data suggest that the novel TCR-targeting LMP1 might allow the potential design of T-cell-based immunotherapeutic strategies against EBV-positive malignancies.

Human Lung Fibroblasts Present Bacterial Antigens to Autologous Lung Th Cells.

Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4+ Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4+ T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4+ T cells isolated from the lung were predominantly (mean 97.5%) CD45RO+ memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4+ T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4+ T cells.

ITOC2 – 031. Immunomodulatory characteristics of Resminostat, a novel HDAC inhibitor in phase II clinical development

Background Anti-cancer effects of HDAC inhibitors were shown to rely on an intact immune system. Additionally, combination of HDAC inhibitors with different cancer immunotherapy approaches was shown to be successful, suggesting that HDAC inhibition itself may engage and mediate an anti-tumoural immune response during cancer therapy. Aim Evaluation of immunomodulating effects of the HDAC inhibitor Resminostat, potentially contributing to its anti-tumoural activity. Method Resminostat effects on mechanisms affecting anti-tumoural immune responses were analysed in hepatocellular HepG2, Huh7, and SNU475 and adenocarcinomic A549 cells. Results Resminostat strongly reduced the expression of immunosuppression mediating enzymes, IDO1 and ARG1. Additionally, Resminostat considerably enhanced the expression of NKG2D ligands, MHC class I molecules, and several cancer testis antigens on several tumour cell lines. Interestingly, Resminostat was also able to up-regulate the expression of MHC class II and co-stimulatory molecule 4-1BBL. Discussion&conclusions Resminostat showed promising immunomodulatory effects demonstrating potential synergism with immunotherapy approaches using opsonizing antibodies (e.g. rituximab), immunostimulating agents (e.g. cytokines, stimulating antibodies, TLR ligands), and immune checkpoint blockers (PD1/PDL1 and CTLA-4 inhibitors).

MHC discrepancies between host tissues and allogeneic NK cells expanded by IL-15 and 4-1BBL ex vivo contribute to GVHD but not GVT (P2150)

Abstract NK cell biology after allogeneic bone marrow transplant (alloBMT) is mainly drawn from studies infusing unmanipulated NK cells. We studied the impact of activating murine NK cells with the co-stimulatory molecule 4-1BBL during expansion with IL-15 ex vivo, and then infused these cells to determine safety and efficacy after alloBMT. NK cells were harvested from Balb/c (H-2d), DBA/2 (H-2d), or CB6F1 (H-2d x H-2b) spleens and cultured with IL-15 in the presence of irradiated artificial APCs transfected with or without 4-1BBL. The addition of 4-1BBL+ cells to IL-15 expanded NK cells decreased yields by up to 50%. The combination of 4-1BBL/IL-15 completely abrogated cytotoxicity against Yac-1 lymphoma, whereas IL-15 alone led to augmented IFNγ production and cytotoxicity. After T cell depleted (TCD) MHC-mismatched alloBMT (CB6F1→Balb/c), recipients of allogeneic NK cell infusions expanded with IL-15/4-1BBL showed weight loss in a dose-dependent fashion and increased lethality from GVHD. Similar results were also seen with CB6F1→C57BL/6. Prior to infusion, IL-15/4-1BBL combination led to less Fas ligand+ NK cells, but after alloBMT, FasL+ NK cells increased. After TCD MHC-matched alloBMT (DBA/2→Balb/c), infusion of NK cells expanded with IL-15 or IL-15/4-1BBL did not cause GVHD, implying MHC discrepancies between donor NK cells and GVHD target tissues contribute to the enhanced alloreactivity induced by 4-1BBL. No differences in graft-versus-tumor effects were observed in vivo.

IL-21 promotes the expansion of CD27+CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells

BackgroundAdoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and −21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma.MethodsWe applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine.ResultsAlthough IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+CD28+ “young” phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs.ConclusionOur data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of “young” effector TIL for ACT.

A novel T cell-based vaccine capable of stimulating long-term functional CTL memory against B16 melanoma via CD40L signaling

The ultimate goal of antitumor vaccines is to develop memory CD8(+) cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. We previously demonstrated that the ovalbumin (OVA)-specific CD4(+) T cell-based (OVA-T(EXO)) vaccine generated using OVA-pulsed dendritic cell (DC(OVA))-released exosomes (EXO(OVA)) stimulate CTL responses via IL-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4(+) OVA-T(EXO) cells or OVA-T(EXO) cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2K(b)/OVA(257-264) tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10(OVA). We demonstrated that CD4(+) OVA-T(EXO) cells stimulated more efficient CTL responses compared to DC(OVA). By assessing primary and recall CTL responses in mice immunized with OVA-T(EXO) or with OVA-T(EXO) lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-T(EXO). Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10(OVA) tumors. Overall, this work suggests that a novel CD4(+) T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination.

Autologous Expanded Natural Killer Cells As a New Therapeutic Option for High-Risk Myeloma

Abstract 2918Natural killer cells (NK) have the unique ability to kill target cells without priming. While their therapeutic potential against various malignancies is becoming more apparent, it has been restricted to the allogeneic setting; NK cells are inhibited by autologous targets by engaging killer immunoglobulin-like receptors with their ligands. Another major challenge to the clinical utility of NK cells is obtaining a sufficient number of NK cells for infusion. Co-culture of blood mononuclear cells (PBMNC) with the leukemic cell line K562, genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BBL (K562mbIL15-41BBL) in the presence of IL2 results in robust expansion and activation of NK cells. To determine if NK cells derived from myeloma (MM) patients can be used therapeutically in the autologous setting, we explored the expansion of NK cells from MM patients, their gene expression profiles (GEP), and their ability to kill autologous and allogeneic MM cells from high-risk patients in vitro and in vivo, and compared these to NK cells from healthy donors (HD).PBMNC from MM patients (N=30) co-cultured with irradiated K562mbIL15-41BBL cells expanded a median of 351 fold (range20–10, 430), comparable to the expansion of HD-derived NK cells (N=15, median 803, range 127–1, 727; p=0.5). GEP of MM non-exp-NK differed from HD non-exp-NK in the expression of only one gene (PRKCi), underexpessed in MM (false discovery rate (FDR) <0.05, p-value <3×10−10). GEP of exp-NK cells from both MM patients and HD was very different from non-exp-NK cells (8 pairs each, 10, 639 differentially overexpressed and 26, 057 underexpressed probe sets, FDR <0.05). Genes associated with proliferation, cytolytic activity, activation, adhesion, migration and cell cycle regulation were highly up-regulated in exp-NK cells. Standard chromium release assays demonstrated that MM exp-NK cells killed both allogeneic and autologous primary MM cells more efficiently compared to non-exp-NK cells, via a perforin mediated mechanism. Blocking studies revealed that the natural cytotoxicity receptors, activating receptors, and DNAX accessory molecule (DNAM-1) played a central role in target cell lysis. The killing ability of MM patient and HD derived exp-NK cells was very similar against allogeneic targets, while primary MM targets were more resistant to killing by autologous exp-NK.The anti-MM activity of allogeneic and autologous exp-NK cells was further examined in vivo. NOD/SCID/IL2R γ-null mice were implanted subcutaneously with a human fetal bone, and primary MM cells or luciferase-transfected OPM2 MM cell line were engrafted into the bone. The tumor burden was determined by ELISA for human Ig and/or bio-imaging. The mice were randomized to control and exp-NK treatment groups. A total of 160 ×106 exp-NK cells, in 4 doses 48 hrs apart, were injected in the exp-NK treatment group via tail vein injection. The mice were administered 1000U of IL2 subcu daily to support the NK cells. The mice were bled on days 7, 14, 21 & 28 for the assessment of human Ig by ELISA and enumerating circulating NK cells by flow cytometry. Exp-NK treated mice had a significantly reduced MM burden by ELISA (p<0.04) on day 21, and exp-NK could be detected in the murine blood up to day 28 post-administration in both primary MM and OPM2 tumor bearing mice. The mice were sacrificed and the tumors were harvested after 4 weeks. A noticeable reduction in tumor burden in the exp-NK cell treated mice was confirmed by histology. NK cells were detected by immunohistochemistry (CD57 or CD16) in the hu-bone implants harvested 28 days after infusion. In conclusion, MM patient-derived NK cells have a similar expansion potential, and MM exp-NK cells have cytolytic activity against allogeneic targets similar to those of HD exp-NK cells, and somewhat reduced activity against autologous targets. These exp-NK cells have significant activity against the aggressive cell line OPM2 and high-risk autologous primary MM cells in vivo. Exp-NK cells trafficked to MM tumors and persisted in the myelomatous hu bone microenvironment for 4 weeks. The anti-MM activity of autologous exp-NK cells is exciting and avails a new therapeutic avenue for patients with GEP-defined high-risk disease. A phase II clinical trial of allogeneic and autologous exp-NK cell therapy for relapsed/refractory high-risk MM is in progress at our institution. Disclosures:No relevant conflicts of interest to declare.

Anti-tumor immune response induced by dendritic cells transduced with truncated PSMA IRES 4-1BBL recombinant adenoviruses

Up-regulation of receptor–ligand pairs during interaction of a peptide-bound MHC complex on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In the present study, we used replication deficient adenoviruses to introduce a tumor-associated Ag (a truncated human prostate-specific membrane antigen (tPSMA)) and the T cell costimulatory molecule 4-1BBL into murine DCs, and observed the ability of these recombinant DCs to elicit tPSMA-directed T-cell responses in vitro and anti-tumor immunity to RM-1-tPSMA in a murine tumor model. Infection of DCs with Ad-tPSMA-IRES-m4-1BBL induced tPSMA-specific proliferative responses and up-regulated CD80 and CD86 s signaling molecules. The cytotoxic T lymphocytes activated by the Ad-tPSMA-IRES-m4-1BBL-transfected DCs showed significantly higher IFN-γ production and cytotoxicity against the RM-1 cells transfected with tPSMA. Moreover, vaccination of mice with Ad-tPSMA-IRES-m4-1BBL-transfected DCs induced a potent protective and therapeutic anti-tumor immunity to RM-1-tPSMA in a tumor model. These results demonstrated that development of DCs engineered to express tPSMA and 4-1BBL by recombinant adenovirus-mediated gene transfer may offer a new strategy for prostate cancer immunotherapy.

Adoptively Transferred Expanded Natural Killer Cells Inhibit Myeloma Tumor Growth In Vivo.

Abstract 953Recently developed culture conditions for expanding and activating natural killer (NK) cells may improve immunotherapeutic options for patients with multiple myeloma. We have previously shown that co-incubation of NK cells from multiple myeloma patients and healthy donors with K562 cells genetically modified to express membrane-bound interleukin 15 and the co-stimulatory molecule 41BBL (K562-mb15-41BBL) leads to a dramatic increase in both NK cell number and anti-myeloma activity in vitro. In this study, we tested the anti-myeloma activity of these expanded NK cells in vivo using a murine model, which supports the growth of myeloma cells in a microenvironment that reproduces that of the human bone marrow. We first implanted human fetal bones subcutaneously into NOD/SCID/IL2Rγ null mice and allowed them to engraft. Luciferase transfected OPM2 myeloma cells were then injected into the human bone fragment. Tumor burden was followed by bio-imaging and ELISA for human Ig. NK cells were expanded from healthy donor PBMC by co-culture with irradiated K562-mb15-41BBL cells in the presence of 300U/ml IL2 for 10-12 days. Myeloma tumor bearing mice were dosed 6-7 days after OPM2 injection (bio-imaging confirmed myeloma engraftment) with expanded NK cells via tail vein injection followed by subcutaneous IL2 to prolong NK cell survival. Flow cytometry was used to track the human NK cells in blood. Histology was performed by H&E staining of formalin-fixed, paraffin embedded tissues harvested at the end of the study. Sufficient NK cells (3×109) for dosing mice were obtained from one unit of healthy donor blood and their ability to kill OPM2 myeloma was confirmed in vitro by chromium release assays. In experiment 1, OPM2-bearing mice received two tail vein injections, 48h apart, comprising PBS, 4 ×107, or 16 ×107 (total dose) expanded NK cells with 100U IL2 given twice per week. We observed significant myeloma growth inhibition in the cohort given 16 ×107 NK cells (p<0.04) and circulating human NK cells could be detected up to day 21 post-administration. Measurement of tumor burden by bio-imaging and ELISA were highly concordant (correlation coefficient = 0.995). Histologic analyses confirmed a dramatic reduction in tumor burden in the mice treated with expanded NK cells and revealed that bone loss was more pronounced in the implanted fetal bones of the control cohort not receiving NK cells. We observed in a subsequent experiment that increasing the IL2 dose to 1000U daily led to in vivo expansion of CSFE-labeled NK cells of up to 10 generations by day 6 post-infusion, which was associated with an enhanced anti-tumor effect in the 16 ×107 dose cohort. Furthermore, we found that multiple injections of NK cells (four injections of 4 ×107 NK cells versus two injections of 8 ×107) were better tolerated in the highest dose cohort (3/3 surviving versus 6/12 surviving). In conclusion, we observed a significant anti-myeloma effect against the aggressive plasma cell leukemia cell line OPM2 with expanded NK cells and low dose IL2. Adjusting the IL2 dose to 1000U daily led to in vivo expansion of NK cells, longer term NK cell persistence, and an increased anti-myeloma effect. Importantly, this IL2 dose is comparable to the 3×106 U daily dose well tolerated in a previous trial with non-expanded NK cells in humans. Single doses of NK cells >8×107 led to high morbidity in this model while multiple doses of 4×107 were well tolerated. Experiments are in progress testing the in vivo effect of expanded NK cells on primary myeloma cell tumors in this model. Our findings support the planned clinical application of ex vivo expanded NK cells. Disclosures:No relevant conflicts of interest to declare.

Prostaglandin E2 enhances T-cell proliferation by inducing the costimulatory molecules OX40L, CD70, and 4-1BBL on dendritic cells

AbstractDendritic cell (DC)–based immunotherapy of malignant diseases relies on 2 critical parameters: antigen transport from the periphery to draining lymph nodes and efficient priming of primary and stimulation of secondary immune responses. Prostaglandin E2 (PGE2) signaling has been shown to be pivotal for DC migration toward lymph node–derived chemokines in vitro and in vivo. Here, we demonstrate that PGE2 induced the expression of the costimulatory molecules OX40L, CD70, and 4-1BBL on human DCs. Short triggering by PGE2 early during DC maturation was sufficient to induce the costimulatory molecules. The expression of the costimulatory molecules was independent of the maturation stimulus but strictly dependent on PGE2 on both monocyte-derived (Mo) DCs and peripheral blood myeloid (PB) DCs. PGE2-matured MoDCs showed enhanced costimulatory capacities resulting in augmented antigen-specific CD4+ and CD8+ T-cell proliferation in primary and recall T-cell responses. Blocking OX40/OX40L signaling impaired the enhanced T-cell proliferation induced by PGE2-matured MoDCs. Moreover, MoDCs matured in the presence of PGE2 induced the expression of OX40, OX40L, and CD70 on T cells facilitating T-cell/T-cell interaction that warrant long-lasting costimulation. This newly identified parameter will help to further optimize DC-based immunotherapy.