Seon Ah Lim

Immunologic and Tissue Biocompatibility of Flexible/Stretchable Electronics and Optoelectronics

Recent development of flexible/stretchable integrated electronic sensors and stimulation systems has the potential to establish an important paradigm for implantable electronic devices, where shapes and mechanical properties are matched to those of biological tissues and organs. Demonstrations of tissue and immune biocompatibility are fundamental requirements for application of such kinds of electronics for long-term use in the body. Here, a comprehensive set of experiments studies biocompatibility on four representative flexible/stretchable device platforms, selected on the basis of their versatility and relevance in clinical usage. The devices include flexible silicon field effect transistors (FETs) on polyimide and stretchable silicon FETs, InGaN light-emitting diodes (LEDs), and AlInGaPAs LEDs, each on low modulus silicone substrates. Direct cytotoxicity measured by exposure of a surrogate fibroblast line and leachable toxicity by minimum essential medium extraction testing reveal that all of these devices are non-cytotoxic. In vivo immunologic and tissue biocompatibility testing in mice indicate no local inflammation or systemic immunologic responses after four weeks of subcutaneous implantation. The results show that these new classes of flexible implantable devices are suitable for introduction into clinical studies as long-term implantable electronics.

Differential expression of immune-associated cancer regulatory genes in low- versus high-dose-rate irradiated AKR/J mice.

AKR/J mice carrying leukemia viral inserts develop thymic lymphoma. Recently, we demonstrated that the incidence of thymic lymphoma was decreased when these mice were raised in a low-dose-rate γ-irradiation facility. In contrast, mice irradiated at a high-dose rate developed severe thymic lymphoma and died much earlier. To understand the genetic changes occurred by low- versus high-dose-rate γ-irradiation whole genome microarray was performed. Both groups of mice demonstrated up-regulation of Ifng, Igbp1, and IL7 in their thymuses, however, mice exposed to high-dose-rate γ-irradiation exhibited marked down-regulation of Sp3, Il15, Traf6, IL2ra, Pik3r1, and Hells. In contrast, low-dose-rate irradiated mice demonstrated up-regulation of Il15 and Jag2. These gene expression profiles imply the impaired immune signaling pathways by high-dose-rate γ-irradiation while the facilitation of anti-tumor immune responses by low-dose-rate γ-irradiation. Therefore, our data delineate common and distinct immune-associated pathways downstream of low- versus high-dose-rate irradiation in the process of cancer progression in AKR/J mice.

Ex Vivo Expansion of Highly Cytotoxic Human NK Cells by Cocultivation with Irradiated Tumor Cells for Adoptive Immunotherapy

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation.

Single Step Isolation and Activation of Primary CD3+ T Lymphocytes Using Alcohol-Dispersed Electrospun Magnetic Nanofibers

Electrospun polymer nanofibers with entrapped magnetic nanoparticles (magnetic NP-NF) represent a novel scaffold substrate that can be functionalized for single-step isolation and activation of specific lymphocyte subsets. Using a surface-embedded T cell receptor ligand/trigger (anti-CD3 monoclonal antibody), we demonstrate, as proof of principle, the use of magnetic NP-NF to specifically isolate, enrich, and activate CD3(+) T cells from a heterogeneous cell mixture, leading to preferential expansion of CD8(+)CD3(+) T cells. The large surface area, adjustable antibody density, and embedded paramagnetic properties of the NP-NF permitted enhanced activation and expansion; its use represents a strategy that is amenable to an efficient selection process for adoptive cellular therapy as well as for the isolation of other cellular subsets for downstream translational applications.

NKG2D ligation relieves 2B4-mediated NK-cell self-tolerance in mice.

Along with MHC class I (MHCI), 2B4 provides nonredundant NK‐cell inhibition in mice. The immunoregulatory role of 2B4 has been increasingly appreciated in models of tumor and viral infection, however, the interactions among 2B4, MHCI, and other activating NK‐cell receptors remain uncertain. Here, we dissect the influence of two distinct inhibitory pathways in modulating NK‐cell‐mediated control of tumors expressing strong activating ligands, including RAE‐1γ. In vitro cytotoxicity and in vivo peritoneal clearance assays using MHCI+CD48+ (RMA‐neo), MHCI+CD48+RAE‐1γ (RMA‐RAE‐1γ), MHCI−CD48+ (RMA‐S‐neo), and MHCI−CD48+RAE‐1γ (RMA‐S‐RAE‐1γ) tumor lines demonstrated that NKG2D activation supersedes the inhibitory effect of both 2B4‐ and MHCI‐mediated immune‐tolerance systems. Furthermore, 2B4KO mice subcutaneously challenged with RMA‐neo and RMA‐S‐neo exhibited reduced tumor growth and significantly prolonged survival compared with WT mice, implying that 2B4 is constitutively engaged in the NK‐cell tolerance mechanism in vivo. Nevertheless, the inhibitory effect of 2B4 is significantly attenuated when NK cells encountered highly stressed tumor cells expressing RAE‐1γ, resulting in an immune response shift toward NK‐cell activation and tumor regression. Therefore, our data highlight the importance of the 2B4‐mediated inhibitory system as an alternate self‐tolerance mechanism, whose role can be modulated by the strength of activating receptor signaling within the tumor microenvironment.

SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity

T cell–based immunotherapy against melanoma-associated antigens can result in on-target/off-tumor cytotoxicity. SLC45A2, a protein overexpressed in melanoma compared with normal melanocytes, was identified as a T-cell target that may be less prone to inducing autoimmune side effects. Cytotoxic T lymphocyte (CTL)–based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient–derived cell lines, we identified a number of shared HLA class I–bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402–restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618–29. ©2017 AACR.

Abstract 122: Hypoxia-enhanced proliferation and effector function of NK cells

Hypoxia promotes tumor cell survival while providing immune cell shaping and resistance. Although NK cells function within hypoxic lymphatics and inflamed tissues, they were shown to mount impaired anti-tumor effector functions in ex-vivo hypoxic environment. This raises a question that additional cues exist in vivo to allow full NK cell activation. By varying the percentage and time of pO 2 exposure ex-vivo, we were able to fine-tune a condition that reverses hypoxia-induced NK cell suppression to become highly proliferative and enhanced cytolytic phenotypes. We found that exposing NK cells to moderate hypoxia in the presence of IL-2 and feeder cells allowed HIF-1α stabilization and its target gene expression, metabolic changes toward glycolysis, specific upregulation of NKp44 receptor, and increased STAT3 phosphorylation with concomitant reduction of apoptosis and p16 senescence pathways. Therefore, the hypoxic exposure allows HIF-1α-mediated adaptation of mature NK cells toward highly proliferative and cytolytic NK phenotypes, which may occur in vivo at the time of tumor growth and inflammation. [This work was supported by the National Research Foundation of Korea (NRF), grants NRF-2013M3A9D3046248] Citation Format: Yunwon Moon, Seon Ah Lim, Kyung-Mi Lee, Hyunsung Park. Hypoxia-enhanced proliferation and effector function of NK 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 122.