Molecular Imaging of PD-1 Unveils Unknown Characteristics of PD-1 Itself by Visualizing "PD-1 Microclusters".

Programmed cell death-1 (PD-1) is one of the most famous coinhibitory receptors that are expressed on effector T cells to regulate their function. The PD-1 ligands, PD-L1 and PD-L2, are expressed by various cells throughout the body at steady state and their expression was further regulated within different pathological conditions such as tumor-bearing and chronic inflammatory diseases. In recent years, immune checkpoint inhibitor (ICI) therapies with anti-PD-1 or anti-PD-L1 has become a standard treatment for various malignancies and has shown remarkable antitumor effects. Since the discovery of PD-1 in 1992, a huge number of studies have been conducted to elucidate the function of PD-1. Herein, this paper provides an overview of PD-1 biological findings and sheds some light on the current technology for molecular imaging of PD-1.

Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters.

With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments.

Requirement of apoptosis-inducing kinase 1 for the induction of bronchial asthma following stimulation with ovalbumin.

BACKGROUND: Bronchial asthma is a chronic inflammatory disease of the airway. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, is activated by environmental stress and plays a crucial role in the induction of apoptosis and inflammation. To examine whether ASK1 is involved in the induction of bronchial asthma, we investigated the role of ASK1 using a genetic approach in the production of cytokines, as well as the development of airway hyperreactivity (AHR) and antibody responses using a murine airway inflammation model. METHODS: ASK1-deficient (ASK1(-/-)) and control wild-type (WT) mice were immunized with ovalbumin (OVA) without alum intraperitoneally, followed by intranasal administration of OVA. Airway infiltration of inflammatory cells, cytokine production, AHR and antibody production were assayed. The asthmatic phenotype was assessed following intranasal administration of IL-13 or TNF-α. RESULTS: ASK1(-/-) mice sensitized with OVA displayed an impaired inflammatory cell infiltration into airways and a decreased AHR relative to WT mice. Moreover, the production of OVA-specific IgE antibodies and proasthmatic cytokines (IL-5, IL-13 and TNF-α) was substantially reduced in OVA-stimulated ASK1(-/-) mice. Intranasal administration of IL-13 and OVA enhanced the accumulation of inflammatory cells in OVA-primed ASK1(-/-) mice. The OVA-induced AHR in response to methacholine was enhanced by IL-13 in WT mice but not ASK1(-/-) mice. CONCLUSIONS: The ASK1 signaling pathway regulates the OVA-induced asthmatic phenotype, specifically AHR sensitivity and cytokine production. Therefore, the ASK1 signaling pathway is a promising target for therapeutic intervention in some asthmatic patients.

Enhanced T cell-independent antibody responses in c-Jun N-terminal kinase 2 (JNK2)-deficient B cells following stimulation with CpG-1826 and anti-IgM.

Although c-Jun NH2-terminal kinase (JNK) 1 and JNK2 have been demonstrated to modulate T cell activation, role of JNKs in B cell activation remains largely unclear. Phosphorylation of JNK2 was increased in murine B cells following stimulation with either anti-IgM or CpG-1826 oligonucleotide (ODN) alone, with a further increase by a combined stimulation with anti-IgM and CpG-1826 ODN. In this study, we examined whether antibody production induced by CpG ODN and/or anti-IgM is affected in B cells from JNK2-deficient (JNK2-/-) mice. After stimulation with CpG ODN or both CpG ODN and anti-IgM, JNK2-/- B cells displayed an enhanced antibody production of IgG1 and IgG2a, with less pronounced in IgG2b production, as assessed by enzyme-linked immunoassay (ELISA). However, IgM production in JNK2-/- B cells by CpG ODN was comparable to that in WT B cells. TLR9 expression was increased in JNK2-/- B cells after stimulation with anti-IgM or both CpG ODN and anti-IgM, suggesting that the anti-IgM/CpG ODN-induced enhancement of antibody production is partly due to the increased expression of TLR9. The enhanced antibody production in JNK2-/- B cells by the combined stimulation does not appear to involve either increased class switch recombination or cell proliferation. Our results provide useful information on the role of JNK2 in antibody responses mediated by T cell-independent antigens.

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin.

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (DeltaPsim) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of DeltaPsim and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H2O2) production with a substantial increase during later time points (36-48 h), accompanied by loss of DeltaPsim and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H2O2 production, loss of DeltaPsim, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-L-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H2O2 production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of DeltaPsim and finally apoptosis.

C-jun N-terminal kinase activation is required for apoptotic cell death induced by TNF-related apoptosis-inducing ligand plus DNA-damaging agents in sarcoma cell lines.

BACKGROUND: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in combination with a chemotherapeutic agent, cis-diammine dichloroplatinum (CDDP) or doxorubicin (DXR), has recently been demonstrated to result in enhanced apoptotic cell death in the sarcoma cell lines MG-63 and SaOS-2. DNA-damaging agents, such as CDDP induced sustained activation of c-Jun N-terminal kinase (JNK), probably leading to apoptosis. In the present study, whether JNK activation is involved in apoptotic cell death induced by combined treatment with CDDP/DXR and TRAIL was addressed. RESULTS: MG-63 or SaOS-2 cells overexpressing the dominant-negative (dn) form of JNK (dnJNK1) were established by transfection with dnJNK1 cDNA. Following stimulation with the chemotherapeutic agent CDDP or TRAIL, both MG-63 and SaOS-2 cells demonstrated enhanced cell death compared with stimulation by either agent alone, as assayed for apoptosis using annexin V staining or mitochondrial membrane potential using DiOC6 staining. Interestingly, partial inhibition of the cell death induced by the combined treatment with CDDP/DXR and TRAIL was found in MG-63 or SaOS-2 cells overexpressing dnJNK1, suggesting that JNK activation is required for the combined treatment. Moreover, induction of caspase-8 activation by TRAIL or TRAIL plus CDDP/DXR was substantially prevented by dnJNK. CONCLUSION: Efficient cell death induced by combined treatment with the chemotherapeutic agents CDDP/DXR and TRAIL is involved in JNK activation in the sarcoma cell lines MG-63 and SaOS-2. These results would be useful for treatment modalities of patients with sarcoma.

Positive modulation of IL-12 signaling by sphingosine kinase 2 associating with the IL-12 receptor beta 1 cytoplasmic region.

IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rbeta1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rbeta1, while the regions including the carboxyl terminus and Box II in the IL-12Rbeta1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-gamma production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-gamma production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rbeta1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.

Requirement of c-Jun N terminal kinase and P38 mitogen-activated protein kinase for anti-IgM-induced reduction in mitochondrial membrane potential in CH31 B lymphoma cells.

CH31 cells have been used for analysis of B cell tolerance, since engagement of membrane immunoglobulin (mIg) results in loss in mitochondrial membrane potential (DeltaPsim), followed by cell death. We have reported that the dominant-negative (dn) form of c-Jun N-terminal kinase (JNK) substantially prevented a loss of DeltaPsim at 24 h, with partial protection around 48 h after anti-IgM stimulation. In this study, we demonstrate that anti-IgM induced a sustained activation of p38 mitogen-activated protein (MAP) kinase. The p38MAP kinase inhibitor SB203580 substantially prevented loss of DeltaPsim at 14 h, with partial prevention (18-24 h) after anti-IgM stimulation. The dnJNK1-mediated prevention of anti-IgM-induced mitochondrial dissipation was enhanced by SB203580 at 42 h, but not 24 h, after stimulation, suggesting that activation of either p38 MAP kinase or JNK may be sufficient for the initiation of early phase of anti-IgM-induced loss of DeltaPsim while both may be necessary in the late phase.