Adoptive infusion of natural killer (NK) cells is being increasingly explored as a therapy in patients with cancer, although clinical responses are thus far limited to patients with hematological malignancies. Inadequate homing of infused NK cells to the tumor site represents a key factor that may explain the poor anti-tumor effect of NK cell therapy against solid tumors. One of the major players in the regulation of lymphocyte chemotaxis is the chemokine receptor chemokine (C-X-C motif) receptor 3 (CXCR3) which is expressed on activated NK cells and induces NK cell migration toward gradients of the chemokine (C-X-C motif) ligand (CXCL9, 10 and 11). Here, we show that ex vivo expansion of human NK cells results in a tenfold increased expression of the CXCR3 receptor compared with resting NK cells (p = 0.04). Consequently, these NK cells displayed an improved migratory capacity toward solid tumors, which was dependent on tumor-derived CXCL10. In xenograft models, adoptively transferred NK cells showed increased migration toward CXCL10-transfected melanoma tumors compared with CXCL10-negative wild-type tumors, resulting in significantly reduced tumor burden and increased survival (median survival 41 vs. 32 days, p = 0.03). Furthermore, administration of interferon-gamma locally in the tumor stimulated the production of CXCL10 in subcutaneous melanoma tumors resulting in increased infiltration of adoptively transferred CXCR3-positive expanded NK cells. Our findings demonstrate the importance of CXCL10-induced chemoattraction in the anti-tumor response of adoptively transferred expanded NK cells against solid melanoma tumors.

Chemokine-driven interactions of immune cells are essential for effective antitumor immunity. Human natural killer (NK) cells can be primed by the interleukin (IL)-1-related proinflammatory cytokine IL-18 for unique helper activity, which promotes dendritic cell (DC) activation and DC-mediated induction of type-1 immune responses against cancer. Here, we show that such IL-18-primed "helper" NK cells produce high levels of the immature DC (iDC)-attracting chemokines CCL3 and CCL4 upon exposure to tumor cells or the additional inflammatory signals IFN-α, IL-15, IL-12, or IL-2. These "helper" NK cells potently attract iDCs in a CCR5-dependent mechanism and induce high DC production of CXCR3 and CCR5 ligands (CXCL9, CXCL10, and CCL5), facilitating the subsequent recruitment of type-1 effector CD8(+) T (Teff) cells. Using cells isolated from the malignant ascites of patients with advanced ovarian cancer, we show that "helper" NK cell-inducing factors can be used to enhance local production of Teff cell-recruiting chemokines. Our findings reveal the unique chemokine expression profile of "helper" NK cells and highlight the potential for using two-signal-activated NK cells to promote homing of type-1 immune effectors to the human tumor environment.

Human leishmaniasis is caused by protozoan Leishmania (L.) parasites and comprises a heterogeneous group of clinical appearances ranging from visceral to cutaneous leishmaniasis. In the New World, L. mexicana mediates American cutaneous leishmaniasis, one of the most common forms of this disease. Two different disease progressions can be observed: (i) self-healing localized cutaneous leishmaniasis (LCL) and (ii) progressive diffuse cutaneous leishmaniasis (DCL). These different forms are associated with a T helper 1 (Th1) or Th2 response, respectively, and are additionally characterized by opposing dermal chemokine profiles. Lesions of LCL show high expression of CCL2/MCP-1, CXCL9/MIG, CXCL10/IP-10 and only low amounts of CCL3/MIP-1alpha. In contrast, lesions of chronic DCL are dominated by the expression of CCL3/MIP-1alpha. This finding implies that CCL2/MCP-1 contributes to the healing process. Indeed, CCL2/MCP-1 induces leishmanicidal activities in human monocytes in contrast to CCL3/MIP-1alpha. This effect is enhanced by interferon-gamma and abrogated by interleukin-4. In the murine model of leishmaniasis, the impact of CCL2/MCP-1 is well documented. Normally resistant mice become susceptible for Leishmania infections if CCR2, the receptor for CCL2/MCP-1, is knocked out. Based on this evidence, we propose that tissue specific expression of these small molecules actively regulates cell traffic and tissue localization of effector cells and, additionally, has direct immunological effects.

Recruitment of circulating natural killer (NK) cells into inflamed lymph nodes is known to provide a potent, IFN-gamma-dependent boost for Th1-polarized immune responses in mouse models. Such NK cell recruitment into draining lymph nodes is induced by certain s.c. injected adjuvants, including mature vaccine dendritic cells (DC), and is mediated by a CXCR3-dependent pathway. Here, we show that monocyte-derived immature human DCs stimulated with polyinosinic acid:polycytidylic acid, IFN-alpha, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IFN-gamma, alpha-type 1-polarized DC (alpha DC1), secrete profuse amounts of the CXCR3 ligand CXCL9/MIG and substantial amounts of CXCL10/IP-10 and CXCL11/I-TAC after withdrawal of maturation stimuli. In sharp contrast, no measurable production of these chemokines was found in DCs after maturation with the current gold standard maturation cocktail for human DC-based cancer vaccines consisting of TNF-alpha, IL-1 beta, IL-6, and prostaglandin-E(2) (PGE(2)-DC). PGE(2)-DCs preferentially produced the Th2 and regulatory T-cell-attracting chemokines CCL17/TARC and CCL22/MDC, whereas only marginal levels of these chemokines were produced by alpha DC1s. Functional studies in vitro showed that supernatants from mature alpha DC1s actively recruited CD3(-)CD56(+) NK cells and that adding anti-CXCL9/MIG antibodies to the alpha DC1 supernatant substantially reduced this recruitment. Finally, alpha DC1s were able to induce IFN-gamma production when cocultured with resting autologous NK cells, but only if concurrent CD40 ligation was provided. These novel findings indicate that injected human alpha DC1-based vaccines have the potential to recruit and activate NK cells during their arrival to draining lymph nodes and that this feature may be of relevance for efficient priming of Th1 cells and CTLs.

The inflammatory chemokines CXCL9, CXCL10, and CXCL11 are predominantly induced by interferon (IFN)-γ and share an exclusive chemokine receptor named CXC chemokine receptor 3 (CXCR3). With a prototype function of directing temporal and spatial migration of activated T cells and natural killer cells, and inhibitory effects on angiogenesis, these CXCR3 ligands have been implicated in infection, acute inflammation, autoinflammation and autoimmunity, as well as in cancer. Intense former research efforts led to recent and ongoing clinical trials using CXCR3 and CXCR3 ligand targeting molecules. Scientific evidence has claimed mutual redundancy, ligand dominance, collaboration or even antagonism, depending on the (patho)physiological context. Most research on their in vivo activity, however, illustrates that CXCL9, CXCL10, and CXCL11 each contribute to the activation and trafficking of CXCR3 expressing cells in a non-redundant manner. When looking into detail, one can unravel a multistep machinery behind final CXCR3 ligand functions. Not only can specific cell types secrete individual CXCR3 interacting chemokines in response to certain stimuli, but also the receptor and glycosaminoglycan interactions, major associated intracellular pathways and susceptibility to processing by particular enzymes, among others, seem ligand-specific. Here, we overview major aspects of the molecular properties and regulatory mechanisms of IFN-induced CXCR3 ligands, and propose that their in vivo non-redundancy is a reflection of the unprecedented degree of versatility that seems inherent to the IFN-related CXCR3 chemokine system.

Chemokines govern leukocyte migration by attracting cells that express their cognate ligands. Many cancer types show altered chemokine secretion profiles, favoring the recruitment of pro-tumorigenic immune cells and preventing the accumulation of anti-tumorigenic effector cells. This can ultimately result in cancer immune evasion. The manipulation of chemokine and chemokine-receptor signaling can reshape the immunological phenotypes within the tumor microenvironment in order to increase the therapeutic efficacy of cancer immunotherapy. Here we discuss the three chemokine-chemokine receptor axes, CXCR1/2-CXCL1-3/5-8, CXCR3-CXCL9/10/11, and CXCR4-CXCL12 and their role on pro-tumorigenic immune cells and anti-tumorigenic effector cells in solid tumors. In particular, we summarize current strategies to target these axes and discuss their potential use in treatment approaches.

Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl(4))-induced liver fibrosis in Cxcr3(-/-) and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet-inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3(-/-) mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF-stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal-regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter-regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo.

CONCLUSIONS

The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis.

Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

Keywords: CXCL9; biomarkers; checkpoint inhibitors; clonal TMB; immunogenicity; immunotherapy; meta-analysis; mutation; neoantigen.

Mesenchymal stem cells (MSCs) have anti-inflammatory and immunosuppressive properties and may be useful in the therapy of diseases such as arteriosclerosis. MSCs have some ability to traffic into inflamed tissues, however to exploit this therapeutically their migratory mechanisms need to be elucidated. This study examines the interaction of murine MSCs (mMSCs) with, and their migration across, murine aortic endothelial cells (MAECs), and the effects of chemokines and shear stress. The interaction of mMSCs with MAECs was examined under physiological flow conditions. mMSCs showed lack of interaction with MAECs under continuous flow. However, when the flow was stopped (for 10 min) and then started, mMSCs adhered and crawled on the endothelial surface, extending fine microvillous processes (filopodia). They then spread extending pseudopodia in multiple directions. CXCL9 significantly enhanced the percentage of mMSCs adhering, crawling and spreading and shear forces markedly stimulated crawling and spreading. CXCL9, CXCL16, CCL20 and CCL25 significantly enhanced transendothelial migration across MAECs. The transmigrated mMSCs had down-regulated receptors CXCR3, CXCR6, CCR6 and CCR9. This study furthers the knowledge of MSC transendothelial migration and the effects of chemokines and shear stress which is of relevance to inflammatory diseases such as arteriosclerosis.

In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis.

The chemokine receptor CXCR3 is highly expressed on Th1 polarized T cells and has been predicted to play an important role in T cell recruitment and immune response in a number of inflammatory and autoimmune diseases. For testing whether CXCR3 plays a role in renal inflammation, CXCR3-deficient mice were generated and nephrotoxic nephritis was induced in C57BL/6 CXCR3(-/-) and C57BL/6 wild-type mice. Induction of the nephrotoxic nephritis leads to an increased renal mRNA expression of IP-10/CXCL10 (8.6-fold), Mig/CXCL9 (2.3-fold), and I-TAC/CXCL11 (4.9-fold) during the autologous phase at days 7 and 14. This increased chemokine expression was paralleled by the renal infiltration of T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Compared with wild-type mice, CXCR3-deficient mice had significantly reduced renal T cell infiltrates. Moreover, CXCR3(-/-) mice developed less severe nephritis, with significantly lower albuminuria, better renal function, and a reduced frequency of glomerular crescent formation. Nephritic wild-type and CXCR3(-/-) mice both elicited an efficient systemic nephritogenic immune response in terms of antigen-specific IgG production and IFN-gamma expression by splenocytes in response to the nephritogenic antigen. These findings indicate that the ameliorated nephritis in CXCR3-deficient mice is due to impaired renal trafficking of effector T cells rather than their inability to mount an efficient humoral or cellular immune response. The neutralization of CXCR3 might be a promising therapeutic strategy for Th1-dependent inflammatory renal disease.

The chemokine receptor CXCR3 is predominantly expressed on T lymphocytes, and its agonists CXCL9, CXCL10 and CXCL11 are IFN-gamma-inducible chemokines that promote Th1 responses. In contrast, the CCR3 agonists CCL11, CCL24 and CCL26 are involved in the recruitment of cells such as eosinophils and basophils during Th2 responses. Here, we report that although CCL11, CCL24 and CCL26 are neither agonists nor antagonists of CXCR3, CCL11 binds with high affinity to CXCR3. This suggests that, in vivo, CXCR3 may act as a decoy receptor, sequestering locally produced CCL11. We also demonstrate that the CXCR3 ligands inhibit CCR3-mediated functional responses of both human eosinophils and CCR3 transfectants induced by all three eotaxins, with CXCL11 being the most efficacious antagonist. The examination of CCR3-CCR1 chimeric constructs revealed that CCL11 and CXCL11 share overlapping binding sites contained within the CCR3 extracellular loops, a region that was previously shown to be essential for effective receptor-activation. Hence, eosinophil responses mediated by chemokines acting at CCR3 may be regulated by two distinct mechanisms: the antagonistic effects of CXCR3 ligands and the sequestration of CCL11 by CXCR3-expressing cells. Such interplay may serve to finely tune inflammatory responses in vivo.

BACKGROUND

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

RESULTS

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2-6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

CONCLUSIONS

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.

BACKGROUND

Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets.

METHODS

We used 24 gastric cancers, 20 Paired normal (PN) and 5 apparently normal gastric tissues obtained from patients with non-gastric cancers (Apparently normal - AN) for the microarray study followed by validation of the significant genes (n = 63) by relative quantitation using Taqman Low Density Array Real Time PCR. We then used a custom made Quantibody protein array to validate the expression of 15 proteins in gastric tissues (4 AN, 9 PN and 9 gastric cancers). The same array format was used to study the plasma levels of these proteins in 58 patients with gastric cancers and 18 from patients with normal/non-malignant gastric conditions.

RESULTS

Seventeen genes (ASPN, CCL15/MIP-1δ, MMP3, SPON2, PRSS2, CCL3, TMEPAI/PMEPAI, SIX3, MFNG, SOSTDC1, SGNE1, SST, IGHA1, AKR1B10, FCGBP, ATP4B, NCAPH2) were shown to be differentially expressed between the tumours and the paired normal, for the first time. EpCAM (p = 0.0001), IL8 (p = 0.0003), CCL4/MIP-1β (p = 0.0026), CCL20/MIP-3α (p = 0.039) and TIMP1 (p = 0.0017) tissue protein levels were significantly different (Mann Whitney U test) between tumours versus AN & PN. In addition, median plasma levels of IL8, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, PDGFR-B and TIMP1 proteins were significantly different between the non-malignant group and the gastric cancer group. The post-surgical levels of EpCAM, IGFBP3, IL8, CXCL10/IP10, CXCL9/MIG, CCL3/MIP-1α, CCL20/MIP-3α, SPP1/OPN and PDGFR-B showed a uniform drop in all the samples studied.

CONCLUSIONS

Our study has identified several genes differentially expressed in gastric cancers, some for the first time. Some of these have been confirmed at the protein level, as well. Some of these proteins will need to be evaluated further for their potential as diagnostic biomarkers in gastric cancers and some could be useful as follow-up markers in gastric cancer.

BACKGROUND

Acute allograft rejection requires a multifaceted immune response involving trafficking of immune cells into the transplant and expression of effector cell functions leading to graft destruction. The chemokine receptor CXCR3 and its ligands, CXCL9, CXCL10 and CXCL11, constitute an important pathway for effector cell recruitment posttransplant. However, analysis of CXCR3 expression and function has been hampered by a general lack of availability of a neutralizing anti-CXCR3 monoclonal antibody (mAb) for use in experimental models.

METHODS

We report the generation, characterization, and use of CXCR3-173, a new hamster mAb specific for mouse CXCR3 that recognizes CXCR3 on cells from wild-type but not CXCR3-/- mice.

RESULTS

Using CXCR3-173 mAb, we demonstrate CXCR3 expression on primary memory phenotype CD4+ and CD8+ T cells, naturally occurring CD4+CD25+ Foxp3+ regulatory T cells, natural killer T cells, and approximately 25% of NK cells. CXCR3-173 blocked chemotaxis in vitro in response to CXCL10 or CXCL11 but not CXCL9. When injected into mice, this mAb significantly prolonged both cardiac and islet allograft survival. When combined with a subtherapeutic regimen of rapamycin, CXCR3-173 mAb induced long-term (>100 day) survival of cardiac and islet allografts. The in vivo effects of CXCR3-173 mAb were not associated with effector lymphocyte depletion.

CONCLUSIONS

These data highlight the utility of CXCR3-173 mAb in developing immunotherapeutic approaches to inhibit transplant rejection and potentially other immune-mediated diseases in murine models.

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial, rate-limiting step of tryptophan (Trp) catabolism along the kynurenine (KYN) pathway, and its induction in cells of the immune system in response to cytokines has been implicated in the regulation of antigen presentation and responses to cell-mediated immune attack. Microarray and quantitative PCR analyses of isolated human islets incubated with interferon (IFN)-gamma for 24 h revealed increased expression of IDO mRNA (>139-fold) and Trp-tRNA synthase (WARS) (>17-fold) along with 975 other transcripts more than threefold, notably the downstream effectors janus kinase (JAK)2, signal transducer and activator of transcription (STAT)1, IFN-gamma regulatory factor-1, and several chemokines (CXCL9/MIG, CXCL10/IP10, CXCL11/1-TAC, CCL2, and CCL5/RANTES) and their receptors. IDO protein expression was upregulated in IFN-gamma-treated islets and accompanied by increased intracellular IDO enzyme activity and the release of KYN into the media. The response to IFN-gamma was countered by interleukin-4 and 1alpha-methyl Trp. Immunohistochemical localization showed IDO to be induced in cells of both endocrine, including pancreatic duodenal homeobox 1-positive beta-cells, and nonendocrine origin. We postulate that in the short term, IDO activation may protect islets from cytotoxic damage, although chronic exposure to various Trp metabolites could equally lead to beta-cell attrition.

CXCR3 is a G-protein-coupled receptor preferentially expressed by activated T cells, NK cells, and dendritic cells. Signaling through gamma interferon-regulated chemokines CXCL9, CXCL10, CXCL11, and CXCR3 plays a critical role in the immune response of many viral pathogens. However, the relevance of CXCR3 for optimal T-cell activation and the induction of regulatory transcription factors (i.e., T-bet and eomesodermin) relative to host immune defense against genital herpes simplex virus type 2 (HSV-2) infection have been poorly defined. In this study, we evaluated the requirement of CXCR3 expression during genital HSV-2 infection using mice deficient in CXCR3 (CXCR3(-/-)) along with wild-type (WT) controls, assessing the resistance of mice to viral infection and focusing on the cytokine/chemokine response, phenotypic analysis of recruited leukocytes, and functional analysis of CD8(+) T cells. CXCR3(-/-) mice showed a heightened sensitivity to infection compared to WT animals in terms of the viral burden in infected tissues as well as elevated mortality. The poor response of CXCR3(-/-) mice to viral infection was associated with reduced cytotoxic T-lymphocyte activity through the impairment of T-bet, perforin, and granzyme B expression by CD8(+) T cells. Corresponding with the defective cytolytic activity, a reduction in recruitment of plasmacytoid dendritic cells and CD80 expression in CD11c(+) dendritic cells in the draining lymph nodes of CXCR3(-/-) mice were detected. Collectively, the results provide a new perspective to CXCR3 signaling for the appropriate activation of CD8(+) T cells required for host defense against genital HSV-2 infection.

IL-18 is involved in the pathogenesis of atopic dermatitis, psoriasis, and allergic contact dermatitis. CXCL9, CXCL10, and CXCL11 recruit type 1 T cells, and the production of these chemokines by keratinocytes is enhanced in these dermatoses. We examined the in vitro effects of IL-18 on IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in human keratinocytes. IL-18 enhanced the IFN-gamma-induced secretion and mRNA expression of CXCL9, CXCL10, and CXCL11 in parallel to the activation of NF-kappaB, STAT1, and IFN-regulatory factor (IRF)-1. Antisense oligonucleotides against NF-kappaB p50, p65, or STAT1 suppressed CXCL9, CXCL10, and CXCL11 production, and antisense IRF-1 suppressed CXCL11 production. Inhibitors of PI3 K, p38 MAPK, and MEK suppressed IL-18 plus IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production and NF-kappaB, STAT1, and IRF-1 activities. IL-18 induced phosphorylation of ERK and Akt, while IFN-gamma induced phosphorylation of p38 MAPK. These results suggest that IL-18 may potentiate IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in keratinocytes by activating NF-kappaB, STAT1, or IRF-1 through PI3 K/Akt and MEK/ERK pathways. These effects of IL-18 may promote the infiltration of type 1 T cells into lesions with inflammatory dermatoses and amplify the skin inflammation. IL-18 may act as a pro-inflammatory cytokine in these dermatoses and thus is a candidate therapeutic target.

Pharyngitis caused by Streptococcus pyogenes is one of the most common bacterial infections in humans and is also a starting point for invasive S. pyogenes infection. Here, we describe that tonsil fluid from patients with streptococcal pharyngitis contains high amounts of the interferon (IFN)-dependent CXC chemokine known as monokine induced by IFN- gamma (MIG)/CXCL9. Also in vitro, inflamed pharyngeal epithelium produced large amounts of MIG/CXCL9 in the presence of bacteria. The CXC chemokines MIG/CXCL9, IFN-inducible protein-10/CXCL10, and IFN-inducible T cell alpha -chemoattractant/CXCL11 all showed antibacterial activity against S. pyogenes, and inhibition of MIG/CXCL9 expression reduced the antibacterial activity at the surface of inflamed pharyngeal cells. S. pyogenes of the clinically important M1 serotype secrets the protein streptococcal inhibitor of complement (SIC), which inhibited the antibacterial activity of the chemokines. As exemplified by S. pyogenes pharyngitis, the data identify a novel innate defense mechanism against invasive bacteria on epithelial surfaces.

UNASSIGNED

Little is known about how the immune microenvironment of breast cancer evolves during disease progression.

UNASSIGNED

We compared tumor infiltrating lymphocyte (TIL) count, PD-L1 protein expression by immunohistochemistry and mRNA levels of 730 immune-related genes using Nanostring technology in primary and metastatic cancer samples.

UNASSIGNED

TIL counts and PD-L1 positivity were significantly lower in metastases. Immune cell metagenes corresponding to CD8, T-helper, T-reg, Cytotoxic T, Dendritic and Mastoid cells, and expression of 13 of 29 immuno-oncology therapeutic targets in clinical development including PD1, PD-L1, and CTLA4 were significantly lower in metastases. There was also coordinated down regulation of chemoattractant ligand/receptor pairs (CCL19/CCR7, CXCL9/CXCR3, IL15/IL15R), interferon regulated genes (STAT1, IRF-1,-4,-7, IFI-27,-35), granzyme/granulysin, MHC class I and immune proteasome (PSMB-8,-9,-10) expression in metastases. Immunotherapy response predictive signatures were also lower. The expression of macrophage markers (CD163, CCL2/CCR2, CSF1/CSFR1, CXCR4/CXCL12), pro-tumorigenic toll like receptor pathway genes (CD14/TLR-1,-2,-4,-5,-6/MyD88), HLA-E, ecto-nuclease CD73/NT5E and inhibitory complement receptors (CD-59,-55,-46) remained high in metastases and represent potential therapeutic targets.

UNASSIGNED

Metastatic breast cancers are immunologically more inert than the corresponding primary tumors but some immune-oncology targets and macrophage and angiogenesis signatures show preserved expression and suggest therapeutic combinations for clinical testing.

OBJECTIVE

Using a combination of clinical, radiographic, functional, and serum protein biomarker assessments, this study was aimed at defining the prevalence and clinical characteristics of interstitial lung disease (ILD) in a large cohort of patients with anti-Jo-1 antibodies.

METHODS

A review of clinical records, pulmonary function test results, and findings on imaging studies determined the existence of ILD in anti-Jo-1 antibody-positive individuals whose data were accumulated in the University of Pittsburgh Myositis Database from 1982 to 2007. Multiplex enzyme-linked immunosorbent assays (ELISAs) for serum inflammation markers, cytokines, chemokines, and matrix metalloproteinases in different patient subgroups were performed to assess the serum proteins associated with anti-Jo-1 antibody-positive ILD.

RESULTS

Among the 90 anti-Jo-1 antibody-positive individuals with sufficient clinical, radiographic, and/or pulmonary function data, 77 (86%) met the criteria for ILD. While computed tomography scans revealed a variety of patterns suggestive of underlying usual interstitial pneumonia (UIP) or nonspecific interstitial pneumonia, a review of the histopathologic abnormalities in a subset of patients undergoing open lung biopsy or transplantation or whose lung tissue was obtained at autopsy (n = 22) demonstrated a preponderance of UIP and diffuse alveolar damage. Analysis by multiplex ELISA yielded statistically significant associations between anti-Jo-1 antibody-positive ILD and elevated serum levels of C-reactive protein (CRP), CXCL9, and CXCL10, which distinguished this disease entity from idiopathic pulmonary fibrosis and anti-signal recognition particle antibody-positive myositis. Recursive partitioning further demonstrated that combinations of these and other serum protein biomarkers can distinguish these disease subgroups at high levels of sensitivity and specificity.

CONCLUSIONS

In this large cohort of anti-Jo-1 antibody-positive individuals, the incidence of ILD approached 90%. Multiplex ELISA demonstrated disease-specific associations between anti-Jo-1 antibody-positive ILD and serum levels of CRP as well as the interferon-gamma-inducible chemokines CXCL9 and CXCL10, highlighting the potential of this approach to define biologically active molecules contributing to the pathogenesis of myositis-associated ILD.

BACKGROUND

Molecular markers based on gene expression profiles have been used in experimental and clinical settings to distinguish cancerous tumors in stage, grade, survival time, metastasis, and drug sensitivity. However, most significant gene markers are unstable (not reproducible) among data sets. We introduce a standardized method for representing cancer markers as 2-level hierarchical feature vectors, with a basic gene level as well as a second level of (more stable) pathway markers, for the purpose of discriminating cancer subtypes. This extends standard gene expression arrays with new pathway-level activation features obtained directly from off-the-shelf gene set enrichment algorithms such as GSEA. Such so-called pathway-based expression arrays are significantly more reproducible across datasets. Such reproducibility will be important for clinical usefulness of genomic markers, and augment currently accepted cancer classification protocols.

RESULTS

The present method produced more stable (reproducible) pathway-based markers for discriminating breast cancer metastasis and ovarian cancer survival time. Between two datasets for breast cancer metastasis, the intersection of standard significant gene biomarkers totaled 7.47% of selected genes, compared to 17.65% using pathway-based markers; the corresponding percentages for ovarian cancer datasets were 20.65% and 33.33% respectively. Three pathways, consisting of Type_1_diabetes mellitus, Cytokine-cytokine_receptor_interaction and Hedgehog_signaling (all previously implicated in cancer), are enriched in both the ovarian long survival and breast non-metastasis groups. In addition, integrating pathway and gene information, we identified five (ID4, ANXA4, CXCL9, MYLK, FBXL7) and six (SQLE, E2F1, PTTG1, TSTA3, BUB1B, MAD2L1) known cancer genes significant for ovarian and breast cancer respectively.

CONCLUSIONS

Standardizing the analysis of genomic data in the process of cancer staging, classification and analysis is important as it has implications for both pre-clinical as well as clinical studies. The paradigm of diagnosis and prediction using pathway-based biomarkers as features can be an important part of the process of biomarker-based cancer analysis, and the resulting canonical (clinically reproducible) biomarkers can be important in standardizing genomic data. We expect that identification of such canonical biomarkers will improve clinical utility of high-throughput datasets for diagnostic and prognostic applications.

OBJECTIVE

Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC).

METHODS

Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma.

RESULTS

Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56(+)CD3(-)CD14(-) NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151-9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added.

CONCLUSIONS

Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.

BACKGROUND

The CXCR3 receptor and its three interferon-inducible ligands (CXCL9, CXCL10 and CXCL11) have been implicated as playing a central role in directing a Th1 inflammatory response. Recent studies strongly support that the CXCR3 receptor is a very attractive therapeutic target for treating autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis and psoriasis, and to prevent transplant rejection. We describe here the in vitro and in vivo pharmacological characterizations of a novel and potent small molecule CXCR3 antagonist, SCH 546738.

RESULTS

In this study, we evaluated in vitro pharmacological properties of SCH 546738 by radioligand receptor binding and human activated T cell chemotaxis assays. In vivo efficacy of SCH 546738 was determined by mouse collagen-induced arthritis, rat and mouse experimental autoimmune encephalomyelitis, and rat cardiac transplantation models. We show that SCH 546738 binds to human CXCR3 with a high affinity of 0.4 nM. In addition, SCH 546738 displaces radiolabeled CXCL10 and CXCL11 from human CXCR3 with IC50 ranging from 0.8 to 2.2 nM in a non-competitive manner. SCH 546738 potently and specifically inhibits CXCR3-mediated chemotaxis in human activated T cells with IC90 about 10 nM. SCH 546738 attenuates the disease development in mouse collagen-induced arthritis model. SCH 546738 also significantly reduces disease severity in rat and mouse experimental autoimmune encephalomyelitis models. Furthermore, SCH 546738 alone achieves dose-dependent prolongation of rat cardiac allograft survival. Most significantly, SCH 546738 in combination with CsA supports permanent engraftment.

CONCLUSIONS

SCH 546738 is a novel, potent and non-competitive small molecule CXCR3 antagonist. It is efficacious in multiple preclinical disease models. These results demonstrate that therapy with CXCR3 antagonists may serve as a new strategy for treatment of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, and to prevent transplant rejection.