PSGL-1 Blockade Induces Classical Activation of Human Tumor-associated Macrophages.

The immune suppressive microenvironment is a major culprit for difficult-to-treat solid cancers. Particularly, inhibitory tumor-associated macrophages (TAM) define the resistant nature of the tumor milieu. To define tumor-enabling mechanisms of TAMs, we analyzed molecular clinical datasets correlating cell surface receptors with the TAM infiltrate. Though P-selectin glycoprotein ligand-1 (PSGL-1) is found on other immune cells and functions as an adhesion molecule, PSGL-1 is highly expressed on TAMs across multiple tumor types. siRNA-mediated knockdown and antibody-mediated inhibition revealed a role for PSGL-1 in maintaining an immune suppressed macrophage state. PSGL-1 knockdown or inhibition enhanced proinflammatory mediator release across assays and donors in vitro. In several syngeneic mouse models, PSGL-1 blockade alone and in combination with PD-1 blockade reduced tumor growth. Using a humanized tumor model, we observed the proinflammatory TAM switch following treatment with an anti-PSGL-1 antibody. In ex vivo patient-derived tumor cultures, a PSGL-1 blocking antibody increased expression of macrophage-derived proinflammatory cytokines, as well as IFNγ, indicative of T-cell activation. Our data demonstrate that PSGL-1 blockade reprograms TAMs, offering a new therapeutic avenue to patients not responding to T-cell immunotherapies, as well as patients with tumors devoid of T cells. SIGNIFICANCE: This work is a significant and actionable advance, as it offers a novel approach to treating patients with cancer who do not respond to T-cell checkpoint inhibitors, as well as to patients with tumors lacking T-cell infiltration. We expect that this mechanism will be applicable in multiple indications characterized by infiltration of TAMs.

Macrophages - Controlling the Bifurcation Between Tumor Existence or Regression.

Macrophages are multifunctional cells that are employed by the tumor to further its growth and adaptation. While tumor-associated macrophages (TAMs) have widely diverse phenotypes, tumors coevolve with the ones that can promote tumorigenesis. Functionally, TAMs/myeloid cells constitute the largest negative influence on the tumor microenvironment and need to be reprogrammed in order to enable successful anti-tumor response in most tumors. It is predicted that successful TAM repolarization has the potential to become a staple of immuno-oncology across most indications.

Characterization of MK-4166, a Clinical Agonistic Antibody That Targets Human GITR and Inhibits the Generation and Suppressive Effects of T Regulatory Cells.

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregsin vitro In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. ©2017 AACR.

Targeting CXCR3 reduces ligand-induced T-cell activation but not development of lung allergic responses.

BACKGROUND: Asthma is a chronic airway inflammatory disease that is associated with a large influx of inflammatory cells. Several chemokines and chemokine receptors play critical roles in the development of allergic airway inflammation. OBJECTIVE: Because polarized human T(H)2 cells express a functional CXCR3 chemokine receptor, we evaluated the effects of a selective CXCR3 inhibitor in a mouse model of allergic airway disease. METHODS: Ovalbumin-specific CD8(+) T effector cells were generated from OT-1 mice in the presence of interleukin 2. The activity of a CXCR3 inhibitor was examined in vitro by monitoring Ca(2+) influx after receptor ligation. In vivo, the activity was assessed in sensitized and challenged mice by monitoring airway function, inflammatory parameters, including cellular infiltrates and cytokines in the bronchoalveolar lavage fluid. RESULTS: Approximately 40% of CD8(+) T effector cells expressed the CXCR3 receptor. In vitro, CXCR3 antagonism reduced Ca(2+) influx after receptor engagement. In contrast, the CXCR3 antagonist had little to no effect on airway function or inflammatory parameters despite adequate exposure levels. CONCLUSIONS: CXCR3 antagonism did not prevent allergen-induced airway hyperresponsiveness or airway inflammation in a mouse allergy model despite having activity in in vitro functional assays.

Drug-induced hemolytic anemia and thrombocytopenia associated with alterations of cell membrane lipids and acanthocyte formation.

CXCR3 is a chemokine receptor, upregulated upon activation of T cells and expressed on nearly 100% of T cells in sites of inflammation. SCH 900875 is a selective CXCR3 receptor antagonist. Thrombocytopenia and severe hemolytic anemia with acanthocytosis occurred in rats at doses of 75, 100, and 150 mg/kg/day. Massively enlarged spleens corresponded histologically to extramedullary hematopoiesis, macrophages, and hemosiderin pigment and sinus congestion. Phagocytosed erythrocytes and platelets were within splenic macrophages. IgG and/or IgM were not detected on erythrocyte and platelet membranes. Ex vivo increased osmotic fragility of RBCs was observed. Lipid analysis of the RBC membrane revealed modifications in phosphatidylcholine, overall cholesterol, and/or sphingomyelin. Platelets exhibited slender filiform processes on their plasma membranes, analogous to those of acanthocytes. The presence of similar morphological abnormalities in acanthocytes and platelets suggests that possibly similar alterations in the lipid composition of the plasma membrane have taken place in both cell types. This phenotype correlated with alterations in plasma lipids (hypercholesterolemia and low triglycerides) that occurred after SCH 900875 administration, although other factors cannot be excluded. The increased cell destruction was considered triggered by alterations in the lipid profile of the plasma membranes of erythrocytes and platelets, as reflected morphologically.

Kinetic assessment and therapeutic modulation of metabolic and inflammatory profiles in mice on a high-fat and cholesterol diet.

The kinetics of metabolic and inflammatory parameters associated with obesity were evaluated in a murine diet-induced obesity (DIO) model using a diet high in fat and cholesterol. Cellular infiltration and mediator production were assessed and shown to be therapeutically modulated by the PPARgamma agonist rosiglitazone. C57BL/6 mice were maintained on a 45% fat/ 0.12% cholesterol (HF/CH) or Chow diet for 3, 6, 16, or 27 weeks. Flow cytometry was employed to monitor peripheral blood monocytes and adipose tissue macrophages (ATM). Gene expression and protein analysis methods were used to evaluate mediator production from total epididymal fat (EF), stromal vascular fraction (SVF), and sorted SVF cells. To investigate therapeutic intervention, mice were fed a HF/CH diet for 12 weeks and then a diet formulated with rosiglitazone (5 mg/kg) for an additional 6 weeks. A HF/CH diet correlated with obesity and a dramatic proinflammatory state. Therapeutic intervention with rosiglitazone attenuated the HF/CH induced inflammation. In addition, a novel population was found that expressed the highest levels of the pro-inflammatory mediators CCL2 and IL-6.

Characterization of a murine keyhole limpet hemocyanin (KLH)-delayed-type hypersensitivity (DTH) model: role for p38 kinase.

Molecular and cellular assessment of dermal delayed-type hypersensitivity (DTH) responses is a useful approach for evaluating the mechanism of action (MOA) of immunomodulatory agents. In the present report, we characterized the delayed-type hypersensitivity response induced by keyhole limpet hemocyanin (KLH), and validated its utility by evaluating an immunomodulator, BIRB-796. Intradermal KLH challenge of the ear pinna following subcutaneous antigen sensitization resulted in a pronounced skin inflammation that peaked at 24-48h. At the molecular level, there was an activation of 3 mitogen-activated protein kinases (MAPKs: p38, JNK and ERK), an induction of the chemokines CCL2/JE, CXCL2/Mip-2, CXCL1/KC, CCL3/Mip-1alpha CCL4/Mip-1beta and CXCL10/IP-10, and expression of the cytokines IL-1beta and IL-10 in the ear parenchyma. Modulation of TNFalpha protein level was only detected in ex-vivo ear whole organ cultures (EWOC). Consistent with this inflammatory profile there was an infiltration of neutrophils and mononuclear cells into the ear parenchyma. BIRB-796, a potent allosteric p38 MAPK inhibitor attenuated the ear swelling response, which correlated with a reduced inflammatory profile. BIRB-796 inhibited p38 but not JNK or ERK kinase activation, decreased multiple chemokines which correlated with a decrease in the infiltration of neutrophils and macrophages; CD4 T cells were modesty reduced. Similarly, there was a decrease of levels of cytokines including IL-1beta, IL-10 and TNFalpha. These data support the utility of this model for evaluating immunomodulators on skin inflammation and suggest that modulation of p38 kinase may be of therapeutic value for the treatment of inflammatory skin conditions.

Nmur1-/- mice are not protected from cutaneous inflammation.

Neuromedin U (Nmu) is a neuropeptide expressed primarily in the gastrointestinal tract and central nervous system. Previous reports have identified two G protein-coupled receptors (designated Nmur1 and Nmur2) that bind Nmu. Recent reports suggest that Nmu mediates immune responses involving mast cells, and Nmur1 has been proposed to mediate these responses. In this study, we generated mice with an Nmur1 deletion and then profiled the responses of these mice in a cutaneous inflammation model utilizing complete Freund's adjuvant (CFA). We report here that mice lacking Nmur1 had normal inflammation responses with moderate changes in serum cytokines compared to Nmur1(+/+) littermates. Although differences in IL-6 were observed in mice lacking Nmu peptide, these mice exhibited a normal response to CFA. Our data argues against a major role for Nmur1 in mediating the reported inflammatory functions of NmU.

Murine CXCR1 is a functional receptor for GCP-2/CXCL6 and interleukin-8/CXCL8.

Functional interleuin-8 (IL-8) receptors (IL-8RA and IL-8RB: CXCR1 and CXCR2, respectively) have been described in human, monkey, dog, rabbit, and guinea pig. Although three IL-8R homologues have been found in rat, only one of these, rat CXCR2, appears to be functional based on responsiveness to ligands. Similarly, CXC chemokines induce biological responses through the murine homolog of CXCR2, but the identification of functional rodent CXCR1 homologues has remained elusive. We have identified and characterized the mouse CXCR1 homologue (mCXCR1). Murine CXCR1 shares 68 and 88% amino acid identity with its human and rat counterparts, respectively. Similar to the tissue distribution pattern of rat CXCR1, we found murine CXCR1 mRNA expression predominantly in lung, stomach, bone marrow, and leukocyte-rich tissues. In contrast to previous reports, we determined that mCXCR1 is a functional receptor. We show predominant engagement of this receptor by mouse GCP-2/CXCL6, human GCP-2, and IL-8/CXCL8 by binding, stimulation of GTPgammaS exchange, and chemotaxis of mCXCR1-transfected cells. Furthermore, murine CXCR1 is not responsive to the human CXCR2 ligands ENA-78/CXCL5, NAP-2/CXCL7, GRO-alpha, -beta, -gamma/CXCL1-3, or rat CINC-1-3. In addition, we show concomitant elevation of mCXCR1 and its proposed major ligand, GCP-2, positively correlated with paw swelling in murine collagen-induced arthritis. This report represents the first description of a functional CXCR1-like receptor in rodents.

Studies to investigate the in vivo therapeutic window of the gamma-secretase inhibitor N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY411,575) in the CRND8 mouse.

Accumulation of amyloid beta-peptide (Abeta) is considered a key step in the etiology of Alzheimer's disease. Abeta is produced by sequential cleavage of the amyloid precursor protein by beta- and gamma-secretase enzymes. Consequently, inhibition of gamma-secretase provides a promising therapeutic approach to treat Alzheimer's disease. Preclinically, several gamma-secretase inhibitors have been shown to reduce plasma and brain Abeta, although they also produce mechanism-based side effects, including thymus atrophy and intestinal goblet cell hyperplasia. The present studies sought to establish an efficient screen for determining the therapeutic window of gamma-secretase inhibitors and to test various means of maximizing this window. Six-day oral administration of the gamma-secretase inhibitor N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-l-alaninamide (LY411,575) reduced cortical Abeta(40) in young (preplaque) transgenic CRND8 mice (ED(50) approximately 0.6 mg/kg) and produced significant thymus atrophy and intestinal goblet cell hyperplasia at higher doses (>3 mg/kg). The therapeutic window was similar after oral and subcutaneous administration and in young and aged CRND8 mice. Both the thymus and intestinal side effects were reversible after a 2-week washout period. Three-week treatment with 1 mg/kg LY411,575 reduced cortical Abeta(40) by 69% without inducing intestinal effects, although a previously unreported change in coat color was observed. These studies demonstrate that the 3- to 5-fold therapeutic window for LY411,575 can be exploited to obtain reduction in Abeta levels without induction of intestinal side effects, that intermittent treatment could be used to mitigate side effects, and that a 6-day dosing paradigm can be used to rapidly determine the therapeutic window of novel gamma-secretase inhibitors.

The human herpes virus 8-encoded chemokine receptor is required for angioproliferation in a murine model of Kaposi's sarcoma.

Kaposi's sarcoma (KS)-associated herpesvirus or human herpes virus 8 is considered the etiological agent of KS, a highly vascularized neoplasm that is the most common tumor affecting HIV/AIDS patients. The KS-associated herpesvirus/human herpes virus 8 open reading frame 74 encodes a constitutively active G protein-coupled receptor known as vGPCR that binds CXC chemokines with high affinity. In this study, we show that conditional transgenic expression of vGPCR by cells of endothelial origin triggers an angiogenic program in vivo, leading to development of an angioproliferative disease that resembles KS. This angiogenic program consists partly in the expression of the angiogenic factors placental growth factor, platelet-derived growth factor B, and inducible NO synthase by the vGPCR-expressing cells. Finally, we show that continued vGPCR expression is essential for progression of the KS-like phenotype and that down-regulation of vGPCR expression results in reduced expression of angiogenic factors and regression of the lesions. Together, these findings implicate vGPCR as a key element in KS pathogenesis and suggest that strategies to block its function may represent a novel approach for the treatment of KS.

IFN-gamma determines distinct clinical outcomes in autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS initiated by autoreactive CD4(+) T cells. EAE classically presents with a progressive ascending paralysis and is a model of multiple sclerosis that recapitulates some aspects of the disease. In this report we describe a mouse strain that spontaneously develops a severe, nonclassical form of EAE with 100% incidence. The distinct clinical phenotype is marked initially by a slight head tilt, progressing to a severe head tilt, spinning, or a rotatory motion. Classical EAE spontaneously occurs in myelin basic protein (MBP)-specific TCR transgenic RAG-1(-/-) mice (referred to as T/R(-)), whereas nonclassical EAE spontaneously occurs in T/R(-) IFN-gamma(-/-) mice (T/R(-)gamma(-)). Thus, the TCR recognizes the same Ag (MBP) and uses identical TCR in both cases. The cellular infiltrate in nonclassical EAE is predominantly found in the brainstem and cerebellum, with very little inflammation in the spinal cord, which is primarily affected in classical disease. Importantly, depending on the genetic makeup and priming conditions of the MBP-specific T cells, nonclassical disease can occur in the presence of an inflammatory infiltrate with eosinophilic, neutrophilic, or monocytic characteristics. Finally, we believe that nonclassical spontaneous EAE could be a useful model for the study of some characteristics of multiple sclerosis not observed in classical EAE, such as the inflammatory responses in the brainstem and cerebellum that can cause vertigo.

Inhibition of intimal hyperplasia in transgenic mice conditionally expressing the chemokine-binding protein M3.

Chemokines have been implicated in the pathogenesis of a wide variety of diseases. This report describes the generation of transgenic mice that conditionally express M3, a herpesvirus protein that binds and inhibits chemokines. In response to doxycycline, M3 expression was induced in a variety of tissues and M3 was detectable in the blood by Western blotting. No gross or histological abnormalities were seen in mice expressing M3. To determine whether M3 expression could modify a significant pathophysiological response, we examined its effect on the development of intimal hyperplasia in response to femoral arterial injury. Intimal hyperplasia is thought to play a critical role in the development of restenosis after percutaneous transluminal coronary angioplasty and in the progression of atherosclerosis. Induction of M3 expression resulted in a 67% reduction in intimal area and a 68% reduction in intimal/medial ratio after femoral artery injury. These data support a role for chemokines in regulating intimal hyperplasia and suggest that M3 may be effective in attenuating this process. This transgenic mouse model should be a valuable tool for investigating the role of chemokines in a variety of pathological states.

Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation.

Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce the pathogenic beta-amyloid (Abeta) peptides, is an attractive approach to the treatment of Alzheimer disease. In addition to APP, however, several other gamma-secretase substrates have been identified (e.g. Notch), and altered processing of these substrates by gamma-secretase inhibitors could lead to unintended biological consequences. To study the in vivo consequences of gamma-secretase inhibition, the gamma-secretase inhibitor LY-411,575 was administered to C57BL/6 and TgCRND8 APP transgenic mice for 15 days. Although most tissues were unaffected, doses of LY-411,575 that inhibited Abeta production had marked effects on lymphocyte development and on the intestine. LY-411,575 decreased overall thymic cellularity and impaired intrathymic differentiation at the CD4(-)CD8(-)CD44(+)CD25(+) precursor stage. No effects on peripheral T cell populations were noted following LY-411,575 treatment, but evidence for the altered maturation of peripheral B cells was observed. In the intestine, LY-411,575 treatment increased goblet cell number and drastically altered tissue morphology. These effects of LY-411,575 were not seen in mice that were administered LY-D, a diastereoisomer of LY-411,575, which is a very weak gamma-secretase inhibitor. These studies show that inhibition of gamma-secretase has the expected benefit of reducing Abeta in a murine model of Alzheimer disease but has potentially undesirable biological effects as well, most likely because of the inhibition of Notch processing.

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice.

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.

Transient lung-specific expression of the chemokine KC improves outcome in invasive aspergillosis.

Invasive aspergillosis is a common and devastating pneumonia in immunocompromised hosts. Neutrophils are critical for defense against this infection, and ELR+ CXC chemokines are potent neutrophil chemoattractants. We hypothesized that transient lung-specific overexpression of one such ligand, KC, in mice with invasive aspergillosis improves the outcome of disease. We generated mice in which transgenic expression of KC was limited to the lungs and occurred only upon exposure to tetracycline analogues, and we exposed them to doxycycline after the onset of invasive aspergillosis. Transgenic mice had a threefold greater survival, a 74% lower lung fungal burden, a greater magnitude of lung KC induction, and an earlier and higher peak of lung neutrophil influx compared with wild-type mice. In addition to a higher number of neutrophils, we found a 1.8-fold higher number of monocytes-macrophages in the lungs of transgenic mice as compared with wild-type mice. Furthermore, transgenic mice had greater lung expression of interferon-gamma and interleukin-12 in response to infection, suggesting that transgenic expression of KC indirectly regulated the expression of other cytokines associated with improved host defense against this pathogen. Taken together, these data suggest that overexpression of KC in the lung in the setting of established invasive aspergillosis results in improved host defense and outcome of disease.

Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in pancreas, but not skin, of transgenic mice.

The CC chemokine CCL21 is a potent chemoattractant for lymphocytes and dendritic cells in vitro. In the murine genome there are multiple copies of CCL21 encoding two CCL21 proteins that differ from each other by one amino acid at position 65 (either a serine or leucine residue). In this report, we examine the expression pattern and biological activities of both forms of CCL21. We found that although both serine and leucine forms are expressed in most tissues examined, the former was the predominant form in lymphoid organs while the latter was predominantly expressed in nonlymphoid organs. When expressed in transgenic pancreas, both forms of CCL21 were capable of inducing the formation of lymph node-like structures composed primarily of T and B cells and a few dendritic cells. Induction of lymph node-like structures by these CCL21 proteins, however, could not be reproduced in every tissue. For instance, no lymphocyte recruitment or accumulation was observed when CCL21 was overexpressed in the skin. We conclude that both forms of CCL21 protein are biologically equivalent in promoting lymphocyte recruitment to the pancreas, and that their ability to induce the formation of lymph node-like structures is dependent on the tissues in which they are expressed.