Jung-Mi Lee

Cutting Edge: Programmed Death-1/Programmed Death Ligand 1 Interaction Regulates the Induction and Maintenance of Invariant NKT Cell Anergy

Invariant NKT (iNKT) cells are a distinct subset of T lymphocytes that recognize glycolipid Ags. Upon TCR stimulation, iNKT cells promptly secrete a wide range of cytokines and therefore have been investigated as a target for immunotherapy. However, after primary activation, iNKT cells become hyporesponsive toward their ligand (anergy). The further mechanism behind iNKT cell anergy is poorly understood. We found that a low level of programmed death-1 (PD-1) was constitutively expressed on iNKT cells and that PD-1 expression was increased after stimulation and lasted at least 2 mo. Moreover, not only did blocking of the PD-1/PD ligand 1 (PD-L1) pathway prevent the induction of anergy in iNKT cells, but anergic iNKT cells also recovered responsiveness and these “rescued” cells efficiently mediated antitumor immunity. Our findings suggest that the PD-1/PD-L1 interaction is essential for the induction and maintenance of iNKT cell anergy.

Immunosuppressive Myeloid-Derived Suppressor Cells Can Be Converted into Immunogenic APCs with the Help of Activated NKT Cells: An Alternative Cell-Based Antitumor Vaccine

Myeloid-derived suppressor cells (MDSCs), which are known to be accumulated in the blood, spleen, and bone marrow of tumor-bearing mice and cancer patients, were tested as APCs for a cellular vaccine because they have phenotypical similarity with inflammatory monocytes and may be differentiated from the same precursors as monocytes. Although MDSCs have immunosuppressive properties, in vivo transferred MDSCs, which present tumor Ag and NKT cell ligand (α-galactosylceramide), significantly prolonged survival time in metastatic tumor-bearing mice in a CD8+ cell-, NK cell-, and NKT cell-dependent manner vs a CD4+ T cell- and host dendritic cell-independent manner. Major concerns about using MDSCs as APCs in a vaccine are their suppression of CTLs and their induction of Foxp3+ regulatory T cells. However, α-galactosylceramide-loaded MDSCs did not suppress CD4+ and CD8+ T cells and allowed for the generation of Ag-specific CTL immunity without increasing the generation of regulatory T cells. Furthermore, stimulation with activated NKT cells induced changes on MDSCs in phenotypical or maturation markers, including CD11b, CD11c, and CD86. Taken together, these findings suggest that NKT cells facilitate the conversion of immunosuppressive MDSCs into immunogenic APCs, eliciting successful antitumor immunity and providing the basis for alternative cell-based vaccines.

The restoration of myeloid-derived suppressor cells as functional antigen-presenting cells by NKT cell help and all-trans-retinoic acid treatment

Myeloid‐derived suppressor cells (MDSCs), which accumulate during tumor progression, have been shown to function as important suppressor cells. In a previous study, we showed that immunosuppressive MDSCs could function as immunogenic antigen‐presenting cells (APCs) with the help of activated natural killer T (NKT) cells. In the current study, however, we found that MDSCs harvested at a late time point after tumor injection (late MDSCs) were poorly immunogenic even when stimulated with activated NKT cells. As tumor growth progressed, the expression of MHC and costimulatory molecules on MDSCs was gradually down‐regulated. Late MDSCs also had innate defects in activation and differentiation mediated by cytokine stimuli. Although late MDSCs treated only with all‐trans‐retinoic acid (ATRA), a stimulating agent for MDSC differentiation, could not become immunogenic, NKT ligand‐loaded, ATRA‐treated late MDSCs could be converted into immunogenic APCs to induce incremental immune responses. Furthermore, these effects were mediated by NKT cells secreting IFNγ, and ATRA‐mediated increases in glutathione (GSH) levels. Thus, combined treatment with differentiating and activating agents is a prerequisite for the conversion of late MDSCs into immunogenic APCs. Collectively, these results suggest that combined treatments are required for the differentiation and activation of late MDSCs in late stage cancer.

Nigrosome 1 Detection at 3T MRI for the Diagnosis of Early-Stage Idiopathic Parkinson Disease: Assessment of Diagnostic Accuracy and Agreement on Imaging Asymmetry and Clinical Laterality

Nigrosomes are calbindin-poor zones within the substantia nigra pars compacta, and are the primary subregion where dopaminergic cells are lost in Parkinson disease. High-resolution 3D multiecho imaging was performed at 3T in 13 healthy subjects and 24 patients with idiopathic Parkinson disease confirmed by 18F-FP-CIT PET. Diagnostic sensitivity, specificity, and accuracy of the nigrosome 1 detection at 3T MR imaging was 100%, 84.6%, and 94.6%, respectively. Further, the clinical laterality was in high concordance with the laterality of the nigrosome 1 detection. BACKGROUND AND PURPOSE: In the early stages of idiopathic Parkinson disease, motor symptoms are usually asymmetric. We aimed to assess the feasibility of nigrosome 1 detection at 3T MR imaging to analyze the agreement of its asymmetry and clinical laterality. MATERIALS AND METHODS: High-resolution 3D multiecho imaging was performed at 3T MR imaging in 13 healthy subjects and 24 patients with idiopathic Parkinson disease confirmed by N-3-fluoropropyl-2-β-carbomethoxy-3-β-(4-iodophenyl) nortropane (18F-FP-CIT) PET. The nigrosome 1 detection findings by using the MR imaging data were rated as “normal,” “possibly abnormal,” and “abnormal” by 2 independent reviewers. The degree of 18F-FP-CIT binding was visually assessed in the caudate nucleus and putamen on PET images. Clinical laterality was evaluated by scores of the Unified Parkinson Disease Rating Scale, Part III. Asymmetry of the affected nigrosome 1 and the degree of 18F-FP-CIT binding were analyzed for agreement with clinical laterality. RESULTS: The diagnostic sensitivity, specificity, and accuracy of the nigrosome 1 detection at 3T MR imaging was 100%, 84.6%, and 94.6%, respectively. Interrater agreements for the abnormality and asymmetry of nigrosome 1 were excellent (κ = 0.863 and 0.835, respectively). In patients with idiopathic Parkinson disease, the agreement of asymmetry between clinical laterality and nigrosome 1 detection was good (κ = 0.724). The degree of the 18F-FP-CIT PET binding showed fair agreement (κ = 0.235) with clinical laterality. CONCLUSIONS: The abnormality involving nigrosome 1 can be detected at 3T MR imaging with an accuracy of 94.6%. The clinical laterality is in high concordance with the laterality of the nigrosome 1 detection at 3T (κ = 0.724).

Functional Changes in Myeloid-Derived Suppressor Cells (MDSCs) during Tumor Growth: FKBP51 Contributes to the Regulation of the Immunosuppressive Function of MDSCs

Myeloid-derived suppressor cells (MDSCs) are increased by tumor-derived factors and suppress anti-tumor immunity. MDSCs obtained at a late time point after tumor injection had stronger suppressive activity than MDSCs obtained at an early time point, as measured by T cell proliferation assays. To find factors in MDSCs that change during tumor growth, we analyzed gene expression profiles from MDSCs at different time points after tumor injection. We found that immune response-related genes were downregulated but protumor function-related genes were upregulated in both monocytic MDSCs (Mo-MDSCs) and polymorphonuclear granulocytic MDSCs (PMN-MDSCs) at the late time point. Among differentially expressed genes, FK506 binding protein 51 (FKBP51), which is a member of the immunophilin protein family and plays a role in immunoregulation, was increased in the Mo-MDSCs and PMN-MDSCs isolated from the late time points. Experiments using small interfering RNA and a chemical inhibitor of FKBP51 revealed that FKBP51 contributes to the regulation of the suppressive function of MDSCs by increasing inducible NO synthase, arginase-1, and reactive oxygen species levels and enhancing NF-κB activity. Collectively, our data suggest that FKBP51 is a novel molecule that can be targeted to regulate the immunosuppressive function of MDSCs.

Tumor Microenvironmental Conversion of Natural Killer Cells into Myeloid-Derived Suppressor Cells

How myeloid-derived suppressor cells (MDSC) emerge in the tumor environment remains unclear. Here, we report that GM-CSF can convert natural killer (NK) cells into MDSCs. When transferred into tumor-bearing mice, adoptively transferred NK cells lost their NK phenotype and were converted into Ly6C(high)Ly6G(high) MDSC. This conversion was abolished by exposure to IL-2 either in vitro or in vivo. Notably, we found that of the 4 maturation stages based on CD11b/CD27 expression levels, only the CD11b(high)CD27(high) NK cells could be converted into CD11b(+)Gr1(+) MDSC ex vivo. Transfer of CD27(high) NK cells from tumor-bearing mice into tumor-bearing recipients was associated with conversion to MDSC in a manner associated with reduced numbers of CD11b(high)CD27(high) and CD11b(high)CD27(low) NK cell populations in the recipients. Our results identify a pathway of MDSC development from immature NK cells in tumor-bearing hosts, providing new insights into how tumor cells modulate their host immune microenvironment to escape immune surveillance.

CD40-targeted recombinant adenovirus significantly enhances the efficacy of antitumor vaccines based on dendritic cells and B cells.

Despite the advantages of using adenoviral vectors for specific antigenic gene delivery in the development of antigen-presenting cell (APC)-based vaccines, the lack of the coxsackievirus-adenovirus receptor (CAR) on APCs limits the use of adenoviral vectors for in vitro gene delivery. In this study, we used a recombinant adapter protein, CFm40L, which consists of the ectodomain of CAR genetically fused to the ectodomain of CD40 ligand (CD40L) via a trimerization motif, to target Her-2/neu- or human papillomavirus 16 (HPV16) E6/E7-encoding adenoviruses to CD40 on dendritic cells (DCs) and B cells. Targeting CD40 enabled the enhancement of tumor antigen delivery and simultaneous activation of APCs via the CD40-CD40L interaction. We found that these transduced DCs and B cells substantially enhanced the CTL response against human Her-2/neu- and HPV16 E6/E7-expressing tumors, resulting in significant inhibition of tumor growth in a murine tumor model. In addition, the use of the CFm40L adapter protein in combination with gemcitabine treatment allowed for a successful immune response against a self-tumor antigen, murine Her-2/neu. Our results suggest that targeting adenovirus to APCs via CD40, using CFm40L, represents a great improvement in anticancer cellular vaccines.

Serum amyloid A3 exacerbates cancer by enhancing the suppressive capacity of myeloid‐derived suppressor cells via TLR2‐dependent STAT3 activation

Myeloid‐derived suppressor cells (MDSCs), which suppress diverse innate and adaptive immune responses and thereby provide an evasion mechanism for tumors, are emerging as a key population linking inflammation to cancer. Although many inflammatory factors that induce MDSCs in the tumor microenvironment are known, the crucial components and the underlying mechanisms remain elusive. In this study, we proposed a novel mechanism by which serum amyloid A3 (SAA3), a well‐known inflammatory factor, connects MDSCs with cancer progression. We found that SAA3 expression in BALB/c mice increased in monocytic MDSCs (Mo MDSCs) with tumor growth. The induction of SAA3 by apo‐SAA treatment in Mo MDSCs enhanced their survival and suppressive activity, while it inhibited GM‐CSF‐induced differentiation. Endogenous SAA3 itself contributed to the increase in the survival and suppressive activity of Mo MDSCs. We demonstrated that SAA3 induced TLR2 signaling, in turn increasing the autocrine secretion of TNF‐α, that led to STAT3 activation. In addition, activated STAT3 enhanced the suppressive activity of Mo MDSCs. Furthermore, SAA3 induction in Mo MDSCs contributed to accelerating tumor progression in vivo. Collectively, these data suggest a novel mechanism by which Mo MDSCs mediate inflammation through SAA3‐TLR2 signaling and thus exacerbate cancer progression by a STAT3‐dependent mechanism.

Retinoic acid alleviates Con A-induced hepatitis and differentially regulates effector production in NKT cells.

Retinoic acid (RA) is a diverse regulator of immune responses. Although RA promotes natural killer T (NKT) cell activation in vitro by increasing CD1d expression on antigen‐presenting cells (APCs), the direct effects of RA on NKT‐cell responses in vivo are not known. In the present study, we demonstrated the effect of RA on the severity of Con A‐induced hepatitis and molecular changes of NKT cells. First, we demonstrated that Con A‐induced liver damage was ameliorated by RA. In correlation with cytokine levels in serum, RA regulated the production of IFN‐γ and IL‐4 but not TNF‐α by NKT cells without influencing the NKT‐cell activation status. However, RA did not alleviate α‐GalCer‐induced liver injury, even though it reduced IFN‐γ and IL‐4 but not TNF‐α levels in serum. This regulation was also detected when liver mononuclear cells (MNCs) or NKT hybridoma cells were treated with RA in vitro. The regulatory effect of RA on NKT cells was mediated by RAR‐α, and RA reduced the phosphorylation of MAPK. These results suggest that RA differentially modulates the production of effector cytokines by NKT cells in hepatitis, and the suppressive effect of RA on hepatitis varies with the pathogenic mechanism of liver injury.

Assessment of dry etching damage in permalloy thin films

Permalloy (Ni81Fe19) thin films are etched by ion beam etching (IBE) or reactive ion etching (RIE), and the variation of magnetic properties with etching time is investigated. Magnetic softness of permalloy thin films deteriorates as etching proceeds, i.e., film thickness decreases. Coercivity begins to increase steeply at a thickness of 0.4 μm and reaches 16 Oe for IBE and 8 Oe for RIE at 0.09 μm. Effective magnetization is increased by 0.2 kG with IBE but decreased by 0.7 kG with RIE. From an atomic force microscopy analysis and independent wet etching experiments, the increase of coercivity is proved to be attributed to the increase of surface roughness as a result of etching. Etching-induced defects are also responsible for the increase of coercivity, which is confirmed by a measurement of electrical resistivity and x-ray diffraction analysis. Ion beam etching process appears to affect magnetic properties to a greater extent than reactive ion etching process.