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.

CCR5-mediated Recruitment of NK Cells to the Kidney Is a Critical Step for Host Defense to Systemic Candida albicans Infection.

C-C chemokine receptor type 5 (CCR5) regulates the trafficking of various immune cells to sites of infection. In this study, we showed that expression of CCR5 and its ligands was rapidly increased in the kidney after systemic Candida albicans infection, and infected CCR5-/- mice exhibited increased mortality and morbidity, indicating that CCR5 contributes to an effective defense mechanism against systemic C. albicans infection. The susceptibility of CCR5-/- mice to C. albicans infection was due to impaired fungal clearance, which in turn resulted in exacerbated renal inflammation and damage. CCR5-mediated recruitment of NK cells to the kidney in response to C. albicans infection was necessary for the anti-microbial activity of neutrophils, the main fungicidal effector cells. Mechanistically, C. albicans induced expression of IL-23 by CD11c+ dendritic cells (DCs). IL-23 in turn augmented the fungicidal activity of neutrophils through GM-CSF production by NK cells. As GM-CSF potentiated production of IL-23 in response to C. albicans, a positive feedback loop formed between NK cells and DCs seemed to function as an amplification point for host defense. Taken together, our results suggest that CCR5-mediated recruitment of NK cells to the site of fungal infection is an important step that underlies innate resistance to systemic C. albicans infection.

Anti-CD137 Suppresses Tumor Growth by Blocking Reverse Signaling by CD137 Ligand.

CD137 (4-1BB) is a T-cell costimulatory molecule, and agonstic CD137 antibodies are currently being evaluated in the clinic as cancer immunotherapy. Recently, it was found that CD137-/- mice or mice injected with agonistic anti-CD137 antibodies exhibit heightened antitumor responses, contrary to expectations based on other knowledge of CD137 function. Here, we report findings related to reverse signaling by CD137 ligand (CD137L) in antigen-presenting dendritic cells (DC) in tumors that address these paradoxical results. Specifically, CD137L suppressed intratumoral differentiation of IL12-producing CD103+ DC and type 1 tumor-associated macrophages (TAM). Differentiation of these cell types is important because they are required to generate IFNγ-producing CD8+ cytotoxic T lymphocytes (Tc1). Notably, CD137L blockade increased levels of IL12 and IFNγ, which promoted intratumoral differentiation of IFNγ-producing Tc1, IL12-producing CD103+ DC, and type 1 TAM within tumors. Our results offer an explanation for the paradoxical effects of CD137 blockade, based on differential immunomodulatory effects of CD137 signaling and reverse signaling in T cells and DC, respectively. Further, they show how CD137L blockade can seed a forward-feedback loop for activation of CD103+ DC/type 1 TAM and Tc1 that can create a self-perpetuating cycle of highly effective immunosurveillance. Cancer Res; 77(21); 5989-6000. ©2017 AACR.

Intratumorally Establishing Type 2 Innate Lymphoid Cells Blocks Tumor Growth.

A long-standing question in the field of tumor immunotherapy is how Th2 cytokines block tumor growth. Their antitumor effects are particularly prominent when they are secreted continuously in tumors, suggesting that Th2 cytokines may create a tumor microenvironment unfavorable for tumor growth independently of adaptive immunity. In this study, we show that local production of IL-33 establishes a high number of type 2 innate lymphoid cells (ILC2s) with potent antitumor activity. IL-33 promotes secretion of a massive amount of CXCR2 ligands from ILC2s but creates a tumor microenvironment where tumor cells express CXCR2 through a dysfunctional angiogenesis/hypoxia/reactive oxygen species axis. These two signaling events converge to reinforce tumor cell-specific apoptosis through CXCR2. Our results identify a previously unrecognized antitumor therapeutic pathway wherein ILC2s play a central role.

Involvement of Protein Kinase C-δ in Vascular Permeability in Acute Lung Injury.

Pulmonary edema is a major cause of mortality due to acute lung injury (ALI). The involvement of protein kinase C-δ (PKC-δ) in ALI has been a controversial topic. Here we investigated PKC-δ function in ALI using PKC-δ knockout (KO) mice and PKC inhibitors. Our results indicated that although the ability to produce proinflammatory mediators in response to LPS injury in PKC-δ KO mice was similar to that of control mice, they showed enhanced recruitment of neutrophils to the lung and more severe pulmonary edema. PKC-δ inhibition promoted barrier dysfunction in an endothelial cell layer in vitro, and administration of a PKC-δ-specific inhibitor significantly increased steady state vascular permeability. A neutrophil transmigration assay indicated that the PKC-δ inhibition increased neutrophil transmigration through an endothelial monolayer. This suggests that PKC-δ inhibition induces structural changes in endothelial cells, allowing extravasation of proteins and neutrophils.

Adjuvant chemotherapy for elderly patients (aged 70 or older) with gastric cancer after a gastrectomy with D2 dissection: A single center experience in Korea.

AIMS: Adjuvant chemotherapy is recommended for gastric cancer after a gastrectomy with D2 dissection. However, its survival benefit in elderly patients is unclear. Here we investigated the use of adjuvant chemotherapy in patients ≥70 years old with stage II or III gastric cancer. METHODS: Patients ≥70 years old diagnosed with stage II or III gastric cancer at Ulsan University Hospital were identified. A retrospective analysis of electronic and paper patient records was performed. RESULTS: From 2008 to 2012, 277 patients ≥70 years old underwent gastrectomy with D2 dissection. Of these patients, 94 were pathologically diagnosed with stage II or III; 55 of these patients (58.5%) received adjuvant chemotherapy and 39 received regular checkups without chemotherapy. Fluoropyrimidine-alone regimens, including TS-1 composed of tegafur, gimestat and otastat potassium (n = 26) and doxifluridine (n = 22), were more commonly used than fluoropyrimidine-platinum combination regimens (n = 7). With a median follow-up of 30.9 (range 0.8-65.5) months, the median relapse-free survival of patients with adjuvant chemotherapy or regular follow-up only was 35.5 and 20.4 months, respectively (P = 0.030). Multivariate analysis revealed that adjuvant chemotherapy is associated with longer relapse-free survival (hazard ratio 0.50; 95% confidence interval 0.27-0.96). There was a trend toward an improved overall survival in the adjuvant chemotherapy group compared with the follow-up only group (P = 0.242). CONCLUSIONS: Although well-designed prospective studies are required, adjuvant chemotherapy may confer a potential survival benefit in elderly patients (aged 70 or older) with stage II or III gastric cancer after a gastrectomy with D2 dissection.

IL-33 Enhances Host Tolerance to Candida albicans Kidney Infections through Induction of IL-13 Production by CD4+ T Cells.

Susceptibility to systemic Candida albicans infection is determined by immune resistance, as well as by the ability to control Candida-induced immunopathologies. We showed previously that exogenous IL-33 can increase resistance to peritoneal C. albicans infection by regulating multiple steps of the neutrophil anti-Candida response. In this study, using a mouse model of systemic candidiasis, we observed that IL-33 administration limited fungal burden and inflammation and increased survival. In kidneys, IL-33 seemed to directly act on neutrophils and CD4(+) T cells: IL-33 administration enhanced fungal clearance by increasing neutrophil phagocytic activity without which Candida proliferation was uncontrollable. In contrast, IL-33 stimulated CD4(+) T cells to produce IL-13, which, in turn, drove the polarization of macrophages toward the M2 type. Furthermore, the absence of IL-13 abolished IL-33-mediated polarization of M2 macrophages and renal functional recovery. In addition, IL-33 and IL-13 acted synergistically to increase M2 macrophage polarization and its phagocytic activity. Overall, this study identifies IL-33 as a cytokine that is able to induce resistance and tolerance and suggests that targeting resistance and tolerance simultaneously with therapeutic IL-33 may benefit patients with systemic candidiasis.

Depletion of Alloreactive T-Cells by Anti-CD137-Saporin Immunotoxin.

Depletion of alloreactive T-lymphocytes from allogeneic bone marrow transplants may prevent graft-versus-host disease (GVHD) without impairing donor cell engraftment, immunity, and the graft-versus-leukemia (GVL) effect. Alloreactive T-cells may be identified by their expression, upon activation, of CD137, a costimulatory receptor and putative surrogate marker for antigen-specific effector T-cells. In this context, we tested the use of anti-CD137-saporin immunotoxin to selectively deplete mouse and human alloreactive T-cells. Anti-CD137 antibodies were internalized by cells within 4 h of binding to the cell surface CD137, and anti-CD137-saporin immunotoxin effectively killed polyclonally activated T-cells or antigen-stimulated T-cells. Transfer of donor T-cells after allodepletion with anti-CD137-saporin immunotoxin failed to induce any evident expression of GVHD; however, a significant GVL effect was observed. Targeting of CD137 with an immunotoxin was also effective in killing polyclonally activated or alloreactive human T-cells. Our results indicate that anti-CD137-saporin immunotoxin may be used to deplete alloreactive T-cells prior to bone marrow transplantation and thereby prevent GVHD and the relapse of leukemia.

IL-33 Priming Enhances Peritoneal Macrophage Activity in Response to Candida albicans.

IL-33 is a member of the IL-1 cytokine family and plays a role in the host defense against bacteria, viruses, and fungi. In this study, we investigated the function of IL-33 and its receptor in in vitro macrophage responses to Candida albicans. Our results demonstrate that pre-sensitization of isolated peritoneal macrophages with IL-33 enhanced their pro-inflammatory cytokine production and phagocytic activity in response to C. albicans. These macrophage activities were entirely dependent on the ST2-MyD88 signaling pathway. In addition, pre-sensitization with IL-33 also increased ROS production and the subsequent killing ability of macrophages following C. albicans challenge. These results indicate that IL-33 may increase anti-fungal activity against Candida through macrophage-mediated resistance mechanisms.

IL-33-induced hematopoietic stem and progenitor cell mobilization depends upon CCR2.

IL-33 has been implicated in the pathogenesis of asthma, atopic allergy, anaphylaxis, and other inflammatory diseases by promoting the production of proinflammatory cytokines and chemokines or Th2 immune responses. In this study, we analyzed the in vivo effect of IL-33 administration. IL-33 markedly promoted myelopoiesis in the bone marrow and myeloid cell emigration. Concomitantly, IL-33 induced hematopoietic stem and progenitor cell (HSPC) mobilization and extramedullary hematopoiesis. HSPC mobilization was mediated mainly through increased levels of CCL7 produced by vascular endothelial cells in response to IL-33. In vivo treatment of IL-33 rapidly induced phosphorylation of ERK, JNK, and p38, and inhibition of these signaling molecules completely blocked the production of CCL7 induced by IL-33. Consistently, inhibitor of CCR2 markedly reduced IL-33-mediated HSPC mobilization in vivo and migration of HSPCs in response to CCL7 in vitro. IL-33-mobilized HSPCs were capable of homing to, and of long-term reconstitution in, the bone marrow of irradiated recipients. Immune cells derived from these recipients had normal antifungal activity. The ability of IL-33 to promote migration of HSPCs and myeloid cells into the periphery and to regulate their antifungal activity represents a previously unrecognized role of IL-33 in innate immunity. These properties of IL-33 have clinical implications in hematopoietic stem cell transplantation.

CD137 in chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) occurs in recipients of allogeneic hematopoietic stem cell transplantation with a high frequency. Preclinical animal chronic GVHD models outlined in this chapter allow for the delineation of events that occur during chronic GVHD development. The DBA/2 → (C56BL/6 × DBA/2)F1 (BDF1) model is characterized by systemic lupus erythematosus (SLE)-like phenotype. The B10.D2 → Balb/c model presents many features of autoimmune scleroderma. The former model is useful in defining how alloreactive donor CD4(+) T cells break B-cell tolerance, whereas the latter model is suitable for dissecting the pathogenesis of organ fibrosis. Our laboratory has demonstrated that injection of a single dose of strong CD137 agonists can prevent or cure chronic GVHD in these two models. In general, these models are particularly suited to screening the immunomodulatory therapeutics.

Inhibition of kidney ischemia-reperfusion injury through local infusion of a TLR2 blocker.

Kidney ischemia-reperfusion injury (IRI) occurs as a result of complex interactions of kidney parenchymal cells and immune cells that are initiated by hypoxic damage of parenchymal cells. In particular, tubular epithelial cells (TECs) not only are susceptible to ischemia but also have an auto-loop system to amplify renal inflammation caused by ischemia and reperfusion. Since endogenous TLR2 ligands released from TECs trigger renal inflammation leading to kidney IRI in an autocrine manner, we hypothesized that local infusion of TLR2 blockers would prevent kidney IRI. In this study, we demonstrated that injection of antagonist anti-TLR2 mAb through the renal vein after cross-clamping significantly reduced the recruitment of NK cells to the kidney after IRI, a phenomenon that is governed by TLR2 signaling in TECs. In addition, intrarenal blocking of TLR2 signaling was shown to inhibit NK cell-mediated neutrophil infiltration and subsequent renal damage. Overall, our simple experiment system will be of help in testing the efficacy of candidate blockers targeting kidney parenchymal cells in inhibition of kidney IRI.

TLR2 signaling in tubular epithelial cells regulates NK cell recruitment in kidney ischemia-reperfusion injury.

Damage-associated molecular patterns released from damaged kidney cells initiate postischemic inflammation, an essential step in the progression of kidney ischemia-reperfusion injury (IRI). However, the mechanism that coordinates this highly specific process in ischemic kidneys remains to be clarified. Previously, we demonstrated that CD137 from NK cells specifically stimulates CD137 ligand (CD137L) on tubular epithelial cells (TECs) such that TECs produced the high CXCR2 chemokine levels required for neutrophil chemotaxis. We report in the present study that endogenous TLR2 ligands released from ischemic TECs induce CCR5 chemokine expression, which is critical to promoting NK cell recruitment. By implanting CD137L(-/-) TECs into the kidney capsule of TLR2(-/-) mice, we further showed that TLR2-mediated NK cell recruitment is an uncoupled event that can occur independently of CD137L signaling in TECs, which is responsible for recruiting neutrophils. Therefore, our findings identify TECs as both a target for kidney damage and also as a master regulator that actively modulates stepwise signaling, leading to the initiation and amplification of acute sterile inflammation that inflicts kidney IRI. Being clinically important, the signaling pathway of innate receptors in epithelial cells may therefore be a good target to block acute sterile inflammation resulting from tissue damage, including kidney IRI.

Interleukin-33: a mediator of inflammation targeting hematopoietic stem and progenitor cells and their progenies.

Inflammation is defined as a physiological response initiated by a variety of conditions that cause insult to the body, such as infection and tissue injury. Inflammation is triggered by specialized receptors in the innate immune system, which recognize microbial components known as pathogen-associated molecular patterns or endogenous signals produced by damaged cells (damage-associated molecular patterns). IL-33 is a cytokine that is released predominantly at the epithelial barrier when it is exposed to pathogens, allergens, or injury-inducing stimuli. IL-33 target cells are various, ranging from hematopoietic stem and progenitor cells (HSPCs) and essentially all types of their progeny to many non-hematopoietic cells. The pleiotropic actions of IL-33 suggest that IL-33 is involved in every phase of the inflammatory process. In this review, we discuss recent advances in the understanding of how IL-33 orchestrates inflammatory responses by regulating HSPCs and innate immune cells.

IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals.

IL-33 is known to play an important role in Th2 immunity. In this study, we investigated the effect of IL-33 pretreatment on anti-fungal response using an acute Candida albicans peritoneal infection model. IL-33 pretreatment induced a rapid fungal clearance and markedly reduced the C. albicans infection-associated mortality. The priming effect of IL-33 occurred during multiple steps of the neutrophil-mediated anti-fungal response. First, the anti-fungal effect occurred due to the rapid and massive recruitment of neutrophils to the site of infection as a result of the release of CXCR2 chemokines by peritoneal macrophages and by reversal of the TLR-induced reduction of CXCR2 expression in neutrophils during IL-33 priming. Second, conditioning of neutrophils by IL-33 activated the TLR and dectin-1 signaling pathways, leading to the upregulation of complement receptor 3 expression induced by C. albicans. Upregulated CR3 in turn increased the phagocytosis of opsonized C. albicans and resulted in the production of high levels of reactive oxygen species and the subsequent enhanced killing activity of neutrophils. Taken together, our results suggest that IL-33 can regulate the anti-fungal activity of neutrophils by collaborative modulation of the signaling pathways of different classes of innate immune receptors.

Sensing endoplasmic reticulum stress by protein kinase RNA-like endoplasmic reticulum kinase promotes adaptive mitochondrial DNA biogenesis and cell survival via heme oxygenase-1/carbon monoxide activity.

Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response, allowing cells to recover folding capacity in the organelle. However, the overwhelming response to severe damage results in apoptotic cell death. Because of the physical proximity between ER and mitochondria, a functional interrelationship between these two organelles, including mitochondrial ATP production and apoptosis, has been suggested. The adaptive response to ER stress includes the maintenance of cellular energetics, which eventually determines cell fate. We previously demonstrated that heme oxygenase-1 (HO-1) activity protects cells against ER stress in a protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent pathway. Here, we provide evidence that PERK-mediated induction of HO-1 in murine macrophages, RAW264.7, relays ER stress to mitochondrial DNA (mtDNA) replication and function. ER stress induced by thapsigargin treatments (10-100 nM) resulted in a 2-fold increase in mtDNA contents compared with that in the untreated control. HO-1 activity on ER stress is proven to be critical for mitochondrial integrity because chemical inhibition (zinc protoporphyrin, 5-20 µM) and genetic depletion of HO-1 by small interference RNA transfection suppress the activation of transcription factors for mitochondrial biogenesis. Carbon monoxide (CO), an enzymatic by-product of HO-1 activity is responsible for the function of HO-1. Limited bioavailability of CO by hemoglobin treatment triggers cell death with a concomitant decline in ATP production. Approximately 78.1% of RAW264.7 cells were damaged in the presence of hemoglobin compared with the percentage of injured cells (26.9%) under ER stress alone. Mitochondrial generation of ATP levels significantly declined when CO availability was limited under prolonged ER stress. Taken together, these results suggest that the cellular HO-1/CO system conveys ER stress to cell survival signals from mitochondria via both the activation of transcriptional factors and functional integrity of mtDNA.

Reverse signaling through the costimulatory ligand CD137L in epithelial cells is essential for natural killer cell-mediated acute tissue inflammation.

Renal ischemia-reperfusion injury (IRI) after kidney transplantation is a major cause of delayed graft function. Even though IRI is recognized as a highly coordinated and specific process, the pathways and mechanisms through which the innate response is activated are poorly understood. In this study, we used a mouse model of acute kidney IRI to examine whether the interactions of costimulatory receptor CD137 and its ligand (CD137L) are involved in the early phase of acute kidney inflammation caused by IRI. We report here that the specific expressions of CD137 on natural killer cells and of CD137L on tubular epithelial cells (TECs) are required for acute kidney IRI. Reverse signaling through CD137L in TECs results in their production of the chemokine (C-X-C motif) receptor 2 ligands CXCL1 and CXCL2 and the subsequent induction of neutrophil recruitment, resulting in a cascade of proinflammatory events during kidney IRI. Our findings identify an innate pathogenic pathway for renal IRI involving the natural killer cell-TEC-neutrophil axis, whereby CD137-CD137L interactions provide the causal contribution of epithelial cell dysregulation to renal IRI. The CD137L reverse signaling pathway in epithelial cells therefore may represent a good target for blocking the initial stage of inflammatory diseases, including renal IRI.

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease.

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.

A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis.

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδin vivo.

A novel method for procuring a large quantity of mature murine eosinophils in vivo.

Eosinophils are rare hematopietic cells that normally constitute only 1~3% of peripheral blood leukocytes. It would be of help for the purpose of research to obtain a large quantity of eosinophils. In this study, we wanted to develop a novel strategy to induce massive expansion of murine eosinophils in vivo, based on the observation showing that treatment of IL-33 induces eosinophilia in mice. We generated an EL-4 lymphoma cell line (herein named EL-4-IL-33) that was engineered to secrete an active form of IL-33. We found that Siglec-F(+) granulocyte numbers increased by 1850-fold in the peritoneal cavity 10 days after inoculation with 1×107 EL-4-IL-33 cells. This number corresponds to 74-fold increase, as compared with the number of Siglec-F(+) granulocytes in mice that received wild-type EL-4 cells. Siglec-F(+) granulocytes expanded by IL-33 had the circular nucleus and expressed eosinophil-specific genes. They also showed some functional characteristics of eosinophils in that they had the ability to respond to IL-5 for survival and eotaxin-1 for chemoattaxis and to produce bioactive eosinophil peroxidase, suggesting that these cells are genuine eosinophils. Our results indicate that sustained secretion of IL-33 by lymphoma cells in the peritoneal cavity is highly effective in increasing peritoneal eosinophil numbers. Therefore, our simple method to obtain eosinphils on a large scale might be of value for eosinophil studies.