Identification of CD137- and CD137L-Expressing Cells in EL-4 Tumor.

The tumor microenvironment (TME) contains noncancerous cells such as various types of immune cells and fibroblasts. Cancer cells direct these stromal cells to create a microenvironment favorable for tumor growth and intercellular interactions have a critical role in this process. In established tumors, interactions between CD137 and its ligand (CD137L) contribute to tumor immune evasion and tumor growth. Therefore, it is important to identify cells expressing CD137 and CD137L within tumors. In this chapter, we will introduce a simple, powerful method of analyzing CD137- and CD137L-expressing tumor cells using Fluorescence-activated cell sorting.

The Murine CD137/CD137 Ligand Signalosome: A Signal Platform Generating Signal Complexity.

CD137, a member of the TNFR family, is a costimulatory receptor, and CD137L, a member of the TNF family, is its ligand. Studies using CD137- and CD137L-deficient mice and antibodies against CD137 and CD137L have revealed the diverse and paradoxical effects of these two proteins in various cancers, autoimmunity, infections, and inflammation. Both their cellular diversity and their spatiotemporal expression patterns indicate that they mediate complex immune responses. This intricacy is further enhanced by the bidirectional signal transduction events that occur when these two proteins interact in various types of immune cells. Here, we review the biology of murine CD137/CD137L, particularly, the complexity of their proximal signaling pathways, and speculate on their roles in immune responses.

The Progress of Investigating the CD137-CD137L Axis as a Potential Target for Systemic Lupus Erythematosus.

Costimulatory molecules facilitate cross-talks among leukocytes via mutual stimulatory and inhibitory signalling, contributing to diverse immunological outcomes in normal physiological responses and pathological conditions. Systemic lupus erythematosus (SLE) is a complex multi-systemic autoimmune condition in which cellular communication through the involvement of costimulatory molecules is crucial in driving proinflammatory responses from the stage of autoantigen presentation to the subsequent process of pathogenic autoantibody production. While the physiology of the costimulatory systems including OX40-OX40L, CD28/CTLA-4-CD80/86, ICOS-B7RP1 and CD70-CD27 has been relatively well studied in SLE, recent data on the immunopathology of the CD137-CD137 ligand (CD137L) system in murine lupus models and patients with SLE highlight the critical role of this costimulatory system in initiating and perpetuating the diverse clinical and serological phenotypes of SLE. CD137, a membrane-bound receptor which belongs to the tumour necrosis factor receptor superfamily, is mainly expressed on activated T cells. Activation of the CD137 receptor via its interaction with CD137L which is expressed on antigen present cells (APC) including B cells, triggers bi-directional signalling; that is, signalling through CD137 as well as signalling through CD137L (reverse signalling), which further activates T cells and polarizes them to the Th1/Tc1 pathway. Further, via reverse CD137L signalling it enhances differentiation and maturation of the APC, particularly of dendritic cells, which subsequently drive proinflammatory cytokine production. In this review, recent data including our experience in the manipulation of CD137L signalling pertaining to the pathophysiology of SLE will be critically reviewed. More in-depth understanding of the biology of the CD137-CD137L co-stimulation system opens an opportunity to identify new prognostic biomarkers and the design of novel therapeutic approaches for advancing the management of SLE.

Deletion of CD137 Ligand Exacerbates Renal and Cutaneous but Alleviates Cerebral Manifestations in Lupus.

The CD137-CD137 ligand (CD137L) costimulatory system is a critical immune checkpoint with pathophysiological implications in autoimmunity. In this study, we investigated the role of CD137L-mediated costimulation on renal, cutaneous and cerebral manifestations in lupus and the underlying immunological mechanism. Lupus-prone C57BL/6lpr-/- (B6.lpr) mice were crossed to C57BL/6.CD137L-/- mice to obtain CD137L-deficient B6.lpr [double knock out (DKO)] mice. We investigated the extent of survival, glomerulonephritis, skin lesions, cerebral demyelination, immune deviation and long-term synaptic plasticity among the two mouse groups. Cytokine levels, frequency of splenic leukocyte subsets and phenotypes were compared between DKO, B6.lpr and B6.WT mice. A 22 month observation of 226 DKO and 137 B6.lpr mice demonstrated significantly more frequent proliferative glomerulonephritis, larger skin lesions and shorter survival in DKO than in B6.lpr mice. Conversely, microglial activation and cerebral demyelination were less pronounced while long-term synaptic plasticity, was superior in DKO mice. Splenic Th17 cells were significantly higher in DKO than in B6.lpr and B6.WT mice while Th1 and Th2 cell frequencies were comparable between DKO and B6.lpr mice. IL-10 and IL-17 expression by T cells was not affected but there were fewer IL-10-producing myeloid (CD11b+) cells, and also lower serum IL-10 levels in DKO than in B6.lpr mice. The absence of CD137L causes an immune deviation toward Th17, fewer IL-10-producing CD11b+ cells and reduced serum IL-10 levels which potentially explain the more severe lupus in DKO mice while leading to reduced microglia activation, lesser cerebral damage and less severe neurological deficits.

CD137 ligand feedback upregulates PD-L1 expression on lung cancer via T cell production of IFN-γ.

BACKGROUND: The expression of PD-L1 and its regulation in tumors remains unclear. The importance of IFN-γ in upregulating the PD-L1 expression in various tumors, and the effects of other essential cytokines in the tumor microenvironment (TME), need to be further elucidated. METHODS: Constitutive expression of PD-L1 and CD137L in all 13 lung cancer cell lines were tested by flow cytometry. CD137L mRNA of lung cancer cell lines was detected by RT-PCR. PD-L1 expression rates following stimulation with these cytokines (IFN-γ, TNFα and IL2) were measured. After coculture of cells expressing CD137L (lung cancer cells or 293FT cells transfected with CD137L plasmid) with T cells, the PDL1 expression of lung cancer cells and IFN-γ in supernatant was detected. RESULTS: Our data revealed that adenocarcinoma and squamous cell carcinoma cells had the highest positive expression rate. IFN-γ was the core-inducing factor for enhancing the PD-L1 expression. CD137L was also widely expressed in the lung cancer cell lines at the mRNA level, whereas its expression was generally low at the protein level. However, the low expression of CD137L protein was still enough to induce T cells to produce IFN-γ, which subsequently increased the PD-L1 expression by lung cancer cells. The CD137 signal induces IFN-γ secretion by T cells, which stimulates high-level of PD-L1 expression in cancer cells; this negative immune regulation may represent a mechanism of immune escape regulation. CONCLUSIONS: CD137L mRNA was widely expressed in lung cancer cell lines whereas levels of protein expression were generally low. The low level of CD137L protein was still enough to induce T cells to produce IFN-γ that subsequently increased PD-L1 expression. The CD137L-induced negative immune regulation may represent a mechanism of immune escape.

Is CD137 Ligand (CD137L) Signaling a Fine Tuner of Immune Responses?

Now, it has been being accepted that reverse signaling through CD137 ligand (CD137L) plays an important role in vivo during hematopoiesis and in immune regulation. However, due to technical difficulty in dissecting both directional signaling events simultaneously in vivo, most biological activities caused by CD137-CD137L interactions are considered as results from signaling events of the CD137 receptor. To make the story more complex, CD137(-/-) and CD137L(-/-) mice have increased or decreased immune responses in a context-dependent manner. In this Mini review, I will try to provide a plausible explanation for how CD137L signaling is controlled during immune responses.

Use of CD137 ligand expression in the detection of small B-cell lymphomas involving the bone marrow.

Staging for small B-cell lymphomas is important for prognostic and therapeutic decision making; however, the detection of lymphoid infiltrates in the bone marrow is often hampered by the lack of specific diagnostic markers. We recently described the hematopoietic tissue distribution patterns of CD137 and CD137 ligand (CD137L), which have shown promise as immunotherapeutic targets. CD137 expression was primarily confined to cells in the microenvironment, whereas CD137L was expressed in neoplastic cells in most B-cell lymphomas. Here we evaluate the use of CD137L in the detection of small B-cell lymphomas involving the bone marrow. To test the potential efficacy of CD137L in detecting bone marrow lymphoid infiltrates, 166 small B-cell lymphomas were evaluated by immunohistochemistry and double-immunofluorescence labeling on formalin-fixed, paraffin-embedded bone marrow core biopsies. CD137L was highly expressed in bone marrows involved by small B-cell lymphomas and included hairy cell leukemia, mantle cell lymphoma, follicular lymphoma, B-lymphoblastic leukemia, and chronic lymphocytic leukemia. In addition, a small subset of marginal zone lymphoma and most of lymphoplasmacytic lymphoma showed staining. Normal bone marrow cells including myeloid, erythroid and megakaryocytic precursors, and reactive lymphoid aggregates lacked staining. Our findings show that immunohistochemistry for CD137L is capable of reliably distinguishing small B-cell lymphomas from reactive lymphoid aggregates. These data also suggest that CD137L is useful in providing staging information for clinical diagnosis and is likely to furnish a potential target for minimal residual disease assessment as well as immunotherapy in patients with stage 4 disease.

The effect of CD137-CD137 ligand interaction on phospholipase C signaling pathway in human endothelial cells.

We previously reported the emerging role of CD137-CD137L interaction in inflammation and atherosclerosis. The mechanism of CD137-CD137L interaction may be related to a variety of signaling pathways. The most important signaling pathway involves the activation of phospholipase C(PLC) which induces the diacylglycerol-protein kinase C(DAG-PKC) and the inositol trisphosphate-intracellular free calcium (IP3-[Ca(2+)]i) pathway. In the current study, we investigated whether CD137-CD137L interaction can stimulate the PLC signaling pathway in human umbilical vein endothelial cells (HUVEC). The diacylglycerol (DAG) and inositol trisphosphate (IP3) levels in HUVEC were measured by radioenzymatic assay. The activity of protein kinase (PKC) was detected by its ability to transfer phosphate from [γ-(32)P]ATP to lysine-rich histone. The [Ca(2+)]i concentrations were measured by flow cytometric analysis. The DAG level and PKC activity were increased in a concentration-dependent, biphasic manner in HUVEC induced by anti-CD137. PKC activity was mainly in the cytosol at rest, and then translocated to the membrane when stimulated by anti-CD137. Similarly, rapid IP3 formation induced by anti-CD137 coincided with the peak of the DAG level. Moreover, anti-CD137 induced peak [Ca(2+)]i responses including the rapid transient phase and the sustained phase. However, anti-CD137L suppressed the activation of the DAG-PKC and IP3-[Ca(2+)]i signaling pathway, which was stimulated by anti-CD137 in HUVEC. In conclusion, the data suggested that CD137-CD137L interaction induces robust activation of the PLC signaling pathway in HUVEC.

Recombinant TAT-CD137 Ligand Cytoplasmic Domain Fusion Protein Induces the Production of IL-6 and TNF-α in Peritoneal Macrophages.

BACKGROUND: The ligand for CD137 (CD137L; also called 4-1BBL) is mainly expressed on activated APCs such as dendritic cells, B cells and macrophages. Even though CD137L functions as a trigger of the CD137 signaling pathway for T cell activation and expansion, engagement of CD137L can deliver a signal leading to the production of proinflammatory cytokines in macrophages. METHODS: We generated cell-permeable TAT-CD137L cytoplasmic domain fusion protein (TAT-CD137Lct) and examined its ability to initiate the CD137L reverse signaling pathway. RESULTS: Treatment of TAT-CD137Lct induced the production of high levels of IL-6 and TNF-α mRNAs and proteins in peritoneal macrophages. TAT-CD137Lct increased phosphorylation of Erk, p38 MAPK and Jnk, and activated transcription factors C/EBP and CREB. However, TAT-CD137Lct did not visibly affect the degradation of the inhibitor of NF-kB (IkBα). We further demonstrated that JNK activation was required for TAT-CD137Lct-induced production of TNF-α, while activation of Erk and p38 MAPK were involved in IL-6 and TNF-α production. CONCLUSION: Our results suggest that TAT-CD137Lct is an effective activator for the CD137L reverse signaling pathway.

CD137-CD137 Ligand Interactions in Inflammation.

The main stream of CD137 studies has been directed to the function of CD137 in CD8(+) T-cell immunity, including its anti-tumor activity, and paradoxically the immunosuppressive activity of CD137, which proves to be of a great therapeutic potential for animal models of a variety of autoimmune and inflammatory diseases. Recent studies, however, add complexes to the biology of CD137. Accumulating is evidence supporting that there exists a bidirectional signal transduction pathway for the CD137 receptor and its ligand (CD137L). CD137/CD137L interactions are involved in the network of hematopoietic and nonhematopoietic cells in addition to the well characterized antigen-presenting cell-T cell interactions. Signaling through CD137L plays a critical role in the differentiation of myeloid cells and their cellular activities, suggesting that CD137L signals trigger and sustain inflammation. The overall consequence might be that the amplified inflammation by CD137L enhances the T-cell activity together with CD137 signals by upregulating costimulatory molecules, MHC molecules, cell adhesion molecules, cytokines, and chemokines. Solving this outstanding issue is urgent and will have an important clinical implication.