John B. Sunwoo

Licensing of natural killer cells by host major histocompatibility complex class I molecules

Self versus non-self discrimination is a central theme in biology from plants to vertebrates, and is particularly relevant for lymphocytes that express receptors capable of recognizing self-tissues and foreign invaders. Comprising the third largest lymphocyte population, natural killer (NK) cells recognize and kill cellular targets and produce pro-inflammatory cytokines. These potentially self-destructive effector functions can be controlled by inhibitory receptors for the polymorphic major histocompatibility complex (MHC) class I molecules that are ubiquitously expressed on target cells. However, inhibitory receptors are not uniformly expressed on NK cells, and are germline-encoded by a set of polymorphic genes that segregate independently from MHC genes. Therefore, how NK-cell self-tolerance arises in vivo is poorly understood. Here we demonstrate that NK cells acquire functional competence through ‘licensing’ by self-MHC molecules. Licensing involves a positive role for MHC-specific inhibitory receptors and requires the cytoplasmic inhibitory motif originally identified in effector responses. This process results in two types of self-tolerant NK cells—licensed or unlicensed—and may provide new insights for exploiting NK cells in immunotherapy. This self-tolerance mechanism may be more broadly applicable within the vertebrate immune system because related germline-encoded inhibitory receptors are widely expressed on other immune cells.

Constitutive activation of transcription factors NF-?B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines

We previously reported that human head and neck squamous cell carcinomas (HNSCCs) express the pro‐inflammatory and pro‐angiogenic cytokines interleukin (IL)‐1α, IL‐6, IL‐8, and granulocyte‐macrophage colony‐stimulating factor in vitro and in vivo. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear factor (NF) κB/Rel A, activator protein‐1 (AP‐1), and CCAAT enhancer‐binding protein β (C/EBPβ, or NF‐IL6), which have been reported to contribute to activation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro‐inflammatory cytokines, by using electrophoretic mobility shift and reporter‐gene assays. Constitutive activation of NF‐κB, AP‐1, and NF‐IL6 DNA‐binding proteins was detected. Supershift analysis with antibodies specific for NF‐κB, AP‐1, and NF‐IL6 binding proteins showed that the NF‐κB–binding protein included p65/Rel A and p50; AP‐1 activity included c‐jun, junB, junD, and Fra‐1; and NF‐IL6 included C/EBPβ. Mutational analysis of the NF‐κB, AP‐1, and NF‐IL6 sites in the IL‐8 promoter region showed that NF‐κB and AP‐1 sites contributed to constitutive IL‐8 reporter activity in HNSCC. HNSCC lines that exhibited increased IL‐8 secretion relative to simian virus 40–immortalized and primary keratinocyte cell lines also demonstrated a concordant increase in NF‐κB reporter activity relative to nonmalignant keratinocytes. We concluded that the early transcription factors NF‐κB, AP‐1, and NF‐IL6 are constitutively activated in human HNSCC cell lines and that NF‐κB and AP‐1 promote expression of the pro‐inflammatory and pro‐angiogenic cytokine IL‐8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and other early gene products that contribute to pathogenesis of the malignant phenotype in HNSCC and in defining potential targets for pharmacologic and molecular therapy of HNSCC. Mol. Carcinog. 26:119–129, 1999. Published 1999 Wiley‐Liss, Inc.

Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-κB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines†

We previously reported that expression of angiogenesis factors interleukin‐8 (IL‐8) and vascular endothelial growth factor (VEGF) is promoted by coactivation of transcription factors nuclear factor‐kappaB (NF‐κB) and activator protein‐1 (AP‐1) by interleukin‐1α in human head and neck squamous cell carcinomas (HNSCC). However, expression of IL‐1 receptor antagonist incompletely blocked reporter gene activity and cytokine expression, suggesting that other upstream signals may contribute to activation. Overexpression and autocrine activation of epidermal growth factor receptor (EGFR) is detected in 90% of HNSCC, and EGFR inhibitors have been reported to inhibit IL‐8 and VEGF expression, but the intermediary signal pathways and transcription factors by which EGFR modulates proangiogenic factors is unknown. EGFR can activate the phosphotidylinositol‐3 kinase (PI3K) and mitogen‐activated/extracellular signal‐regulated kinase (MEK) pathways, which can potentially modulate activation of NF‐κB and AP‐1, respectively. In our study, we examined the effect of EGF and antagonists of EGFR, PI3K and MEK on NF‐κB and AP‐1 activation and IL‐8 and VEGF expression in HNSCC cell lines UM‐SCC‐9 and 11B in which EGFR is overexpressed and activated. Recombinant EGF induced EGFR phosphorylation, activation of NF‐κB and AP‐1 reporter genes and IL‐8 and VEGF expression, indicating that EGFR can mediate coactivation of both transcription factors and cytokine genes in HNSCC. EGFR antagonist PD153035 and anti‐EGFR antibody C225 completely inhibited EGF‐induced reporter activity and cytokine expression, but only partially inhibited constitutive activity. MEK inhibitor U0126 preferentially blocked AP‐1 activity and expression of both IL‐8 and VEGF, while PI3K inhibitor LY‐294002 or a dominant negative inhibitor‐κB preferentially blocked NF‐κB activation and expression of IL‐8 but not VEGF. EGFR, PI3K and MEK antagonists inhibited growth of HNSCC. We conclude that antagonists of EGFR, PI3K and MEK signal pathways have inhibitory activity against EGFR‐induced NF‐κB and AP‐1 activation, IL‐8 and VEGF expression and growth by HNSCC. Published 2002 Wiley‐Liss, Inc.

HLA alleles determine differences in human natural killer cell responsiveness and potency

Epidemiological studies have associated certain human disease outcomes with particular killer cell Ig-like receptor (KIR) and HLA genotypes. However, the functional explanation for these associations is poorly understood, because the KIRs were initially described as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. Yet resolution of infections is often associated with genotypic pairing of inhibitory KIRs with their cognate HLA ligands. Recent studies in mice indicate a second role for MHC-specific inhibitory receptors, i.e., self-MHC recognition confers functional competence on the NK cell to be triggered through their activation receptors, a process termed licensing. As a result, licensed NK cells with self-MHC-specific receptors are more readily activated as compared with unlicensed NK cells without self-MHC-specific receptors. Such results predict that human NK cells may undergo a similar process. Here, we examined the human NK cell subset expressing KIR3DL1, the only known KIR specific for HLA-Bw4 alleles. The KIR3DL1+ subset in normal donors with two HLA-B-Bw4 genes displayed increased responsiveness to tumor stimulation compared with the KIR3DL1+ subset from individuals with only one or no Bw4 genes. By contrast, NK cells lacking KIR3DL1 showed no differences. Therefore, these data indicate that particular KIR and HLA alleles are associated with more responsive NK cells, strongly suggesting that human NK cells are also subjected to NK cell licensing, and providing a potential functional explanation for the influence of KIR and HLA genes in disease as well as interindividual differences in NK cell potency.

Nuclear Factor-κB is an Important Modulator of the Altered Gene Expression Profile and Malignant Phenotype in Squamous Cell Carcinoma

We reported previously that transcription factor nuclear factor (NF)-κB is constitutively activated in human and murine squamous cell carcinomas (SCCs). The role of NF-κB in the cumulative changes in gene expression with transformation and progression of the murine SCC Pam 212 and after switching off NF-κB by a dominant negative inhibitor κB mutant (IκBαM) was explored by profiling with a 15,000-element cDNA micoarrray. Remarkably, NF-κB modulated the expression of >60% of the 308 genes differentially expressed between normal keratinocytes and metastatic SCCs. NF-κB directly or indirectly modulated expression of programs of genes functionally linked to proliferation, apoptosis, adhesion, and angiogenesis. Among these, changes in expression of cyclin D1, inhibitor of apoptosis-1, mutant Trp53, and β-catenin detected with modulation of NF-κB by microarray were confirmed by Western and Northern blot. NF-κB DNA binding motifs were detected in the promoter of ∼63% of genes showing increased expression and 33% of the genes showing decreased expression. The ACTACAG motif implicated in the NF-κB-dependent down-regulation of mRNA expression of MyoD and Sox9 was detected in the coding portion of about 15% of genes showing increased or decreased expression. Inactivation of NF-κB inhibited malignant phenotypic features including proliferation, cell survival, migration, angiogenesis, and tumorigenesis. These results provide evidence that NF-κB is an important modulator of gene expression programs that contribute to the malignant phenotype of SCC.

Targeting CD137 enhances the efficacy of cetuximab

Treatment with cetuximab, an EGFR-targeting IgG1 mAb, results in beneficial, yet limited, clinical improvement for patients with head and neck (HN) cancer as well as colorectal cancer (CRC) patients with WT KRAS tumors. Antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells contributes to the efficacy of cetuximab. The costimulatory molecule CD137 (4-1BB) is expressed following NK and memory T cell activation. We found that isolated human NK cells substantially increased expression of CD137 when exposed to cetuximab-coated, EGFR-expressing HN and CRC cell lines. Furthermore, activation of CD137 with an agonistic mAb enhanced NK cell degranulation and cytotoxicity. In multiple murine xenograft models, including EGFR-expressing cancer cells, HN cells, and KRAS-WT and KRAS-mutant CRC, combined cetuximab and anti-CD137 mAb administration was synergistic and led to complete tumor resolution and prolonged survival, which was dependent on the presence of NK cells. In patients receiving cetuximab, the level of CD137 on circulating and intratumoral NK cells was dependent on postcetuximab time and host FcyRIIIa polymorphism. Interestingly, the increase in CD137-expressing NK cells directly correlated to an increase in EGFR-specific CD8+ T cells. These results support development of a sequential antibody approach against EGFR-expressing malignancies that first targets the tumor and then the host immune system.

ERK1/2 regulation of CD44 modulates oral cancer aggressiveness

Carcinogen-induced oral cavity squamous cell carcinoma (OSCC) incurs significant morbidity and mortality and constitutes a global health challenge. To gain further insight into this disease, we generated cell line models from 7,12-dimethylbenz(a)anthracene-induced murine primary OSCC capable of tumor formation upon transplantation into immunocompetent wild-type mice. Whereas several cell lines grew rapidly and were capable of metastasis, some grew slowly and did not metastasize. Aggressively growing cell lines displayed ERK1/2 activation, which stimulated expression of CD44, a marker associated with epithelial to mesenchymal transition and putative cancer stem cells. MEK (MAP/ERK kinase) inhibition upstream of ERK1/2 decreased CD44 expression and promoter activity and reduced cell migration and invasion. Conversely, MEK1 activation enhanced CD44 expression and promoter activity, whereas CD44 attenuation reduced in vitro migration and in vivo tumor formation. Extending these findings to freshly resected human OSCC, we confirmed a strict relationship between ERK1/2 phosphorylation and CD44 expression. In summary, our findings identify CD44 as a critical target of ERK1/2 in promoting tumor aggressiveness and offer a preclinical proof-of-concept to target this pathway as a strategy to treat head and neck cancer.

Concurrent Paclitaxel and Radiation in the Treatment of Locally Advanced Head and Neck Cancer

PURPOSE To determine the feasibility of an organ preservation regimen consisting of infusional paclitaxel administered concurrently with radiotherapy to patients with locally advanced head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS Thirty-three previously untreated patients with stage III or IV tumors were enrolled onto the study. Paclitaxel was administered as a 120-hour continuous infusion every 3 weeks during the course of radiation therapy. Sixteen patients received a paclitaxel dose of 105 mg/m(2), and 17 patients received 120 mg/m(2). Radiation was delivered in a standard format at 1.8 Gy/d to a total dose of 70.2 to 72 Gy. RESULTS Three months after therapy, a 76% complete response (CR) at the primary site and a 70% overall CR was achieved. At 36 months, locoregional control was 55.7%, overall survival was 57.8%, and disease-free survival was 51.1%. The median survival duration for all 33 patients was greater than 50 months at the time of this report. Local toxicities including mucositis, dysphagia, and skin reactions were severe but tolerable. All patients retained functional speech, and all but four patients were swallowing food 3 months after treatment. Steady-state plasma concentrations for paclitaxel were not achieved during a 120-hour infusion, suggesting a nonlinear process. Tumor volume quantified by pretreatment computerized tomography imaging was associated with likelihood of response and survival. CONCLUSION Paclitaxel administered as a 120-hour continuous infusion in combination with radiotherapy is a feasible and promising treatment for patients with advanced HNSCC.

Identification of human NK cells that are deficient for signaling adaptor FcRγ and specialized for antibody-dependent immune functions

NK cells respond to tumor and virus-infected cells directly through several activation receptors, including natural cytotoxicity receptors, or indirectly through the activating Fc receptor CD16 for antibody-coated cells. Triggering of NK-cell effector functions through these receptors depends on physically associated transmembrane signaling adaptors, such as FcRγ (also known as FcεRIγ) and CD3ζ, both of which have been traditionally believed to be expressed by all mature NK cells. However, we have identified a distinct subset of human NK cells that are deficient for FcRγ expression but express normal levels of CD3ζ. FcRγ-deficient NK cells were readily detectable in about one-third of the healthy individuals examined. The deficiency was confined to the CD56(dim) population and was due to low FcRγ mRNA. FcRγ-deficient NK cells displayed dramatically reduced expression of the natural cytotoxicity receptors NKp46 and NKp30 but still expressed substantial levels of CD16. Compared to FcRγ-expressing NK cells, FcRγ-deficient NK cells showed poor direct reactivity toward tumor targets as measured by cytokine production and degranulation. Unexpectedly, however, FcRγ-deficient NK cells exhibited significantly more robust responsiveness upon stimulation through CD16, particularly for cytokine production, compared to FcRγ-expressing NK cells. Thus, our study reveals FcRγ-deficient NK cells as a novel subset of human NK cells that have remarkably potent responses toward antibody-coated targets. These findings also illustrate a differential contribution of FcRγ and CD3ζ for the expression and functional activity of their associated receptors.

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon receptor

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its role in the differentiation and activity of immune cell subsets; however, its role in regulating the activity of natural killer (NK) cells has not been described. Here, we show that AhR expression is induced in murine NK cells upon cytokine stimulation. We show that in the absence of AhR, NK cells have reduced cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo, despite having normal development and maturation markers. Although AhR was first identified to bind the xenobiotic compound dioxin, AhR is now known to bind a variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that activation of AhR with an endogenous tryptophan derivative, 6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner. Finally, similar effects on NK cell potency occur with AhR dietary ligands, potentially explaining the numerous associations that have been observed in the past between diet and NK cell function. Our studies introduce AhR as another regulator of NK cell activity in vivo.