Clinical performance of a prefabricated immunofluorescence assay for nasopharyngeal cancer screening.

BACKGROUND: Epstein-Barr virus (EBV) IgA serology for viral capsid antigen (VCA) and early antigen (EA) aids early detection of nasopharyngeal cancer (NPC), resulting in improved survival. We evaluated the diagnostic performance of a prefabricated immunofluorescent assay (IFA) for NPC screening in high-risk individuals. METHODS: Sera from 96 biopsy-proven patients with NPC diagnosed at the outpatient clinic and 96 healthy family members were tested for EBV-VCA IgA and EBV-EA IgA using the prefabricated IFA from EUROIMMUN (EI) and the traditional immunofluorescence method. RESULTS: The AUC of EI EBV-VCA IgA and EBV-EA IgA was 0.907 (95% confidence interval [CI]: 0.894-0.965) and 0.898 (95% CI: 0.848-0.947), respectively. Combined testing with the prefabricated assay at a threshold of VCA ≥1:320 or EA ≥1:10 showed 92.7% sensitivity and 81.2% specificity. Overall, the traditional EBV-EA IgA assay demonstrated the best accuracy (sensitivity 91.7% and specificity 96.9%) at a threshold of ≥1:5. CONCLUSION: While the traditional IFA method was more accurate, the prefabricated IFA test kit can be a useful tool for NPC screening in high-risk populations.

Deep Learning Fuzzy Inference: An Interpretable Model for Detecting Indirect Immunofluorescence Patterns Associated with Nasopharyngeal Cancer.

The detection of serum Epstein-Barr virus antibodies by immunofluorescence assay (IFA) is considered the gold standard screening test for nasopharyngeal cancer (NPC) in high-risk populations. Given the high survival rate after early detection in asymptomatic patients, compared to the poor prognosis in patients with late-stage NPC, screening using IFA has tremendous potential for saving lives in the general population. However, IFA requires visual interpretation of cellular staining patterns by trained pathology staff, making it labor intensive and hence nonscalable. In this study, an automated fuzzy inference (FI) system achieved high agreement with a human IFA expert in identifying cellular patterns associated with NPC (κ = 0.82). The integration of a deep learning module into FI further improved the performance of FI (κ = 0.90) and reduced the number of uncertain cases that required manual evaluation. The performance of the resulting hybrid model, termed deep learning FI (DeLFI), was then evaluated with a separate testing set of clinical samples. In this clinical validation, DeLFI outperformed human evaluation on the area under the curve (0.926 versus 0.821) and closely matched human performance on Youden J index (0.81 versus 0.80). Data from this study indicate that the combination of deep learning with FI in DeLFI has the potential to improve the scalability and accuracy of NPC detection.

TCR-like antibodies mediate complement and antibody-dependent cellular cytotoxicity against Epstein-Barr virus-transformed B lymphoblastoid cells expressing different HLA-A*02 microvariants.

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV-transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.

Increasing the accuracy and scalability of the Immunofluorescence Assay for Epstein Barr Virus by inferring continuous titers from a single sample dilution.

High Epstein Barr Virus (EBV) titers detected by the indirect Immunofluorescence Assay (IFA) are a reliable predictor of Nasopharyngeal Carcinoma (NPC). Despite being the gold standard for serological detection of NPC, the IFA is limited by scaling bottlenecks. Specifically, 5 serial dilutions of each patient sample must be prepared and visually matched by an evaluator to one of 5 discrete titers. Here, we describe a simple method for inferring continuous EBV titers from IFA images acquired from NPC-positive patient sera using only a single sample dilution. In the first part of our study, 2 blinded evaluators used a set of reference titer standards to perform independent re-evaluations of historical samples with known titers. Besides exhibiting high inter-evaluator agreement, both evaluators were also in high concordance with historical titers, thus validating the accuracy of the reference titer standards. In the second part of the study, the reference titer standards were IFA-processed and assigned an 'EBV Score' using image analysis. A log-linear relationship between titers and EBV Score was observed. This relationship was preserved even when images were acquired and analyzed 3days post-IFA. We conclude that image analysis of IFA-processed samples can be used to infer a continuous EBV titer with just a single dilution of NPC-positive patient sera. This work opens new possibilities for improving the accuracy and scalability of IFA in the context of clinical screening.

Targeting Epstein-Barr virus-transformed B lymphoblastoid cells using antibodies with T-cell receptor-like specificities.

Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases.

The Role of Epstein-Barr Virus DNA Load and Serology as Screening Tools for Nasopharyngeal Carcinoma.

OBJECTIVE: Screening for nasopharyngeal carcinoma (NPC) among family members has been advocated in endemic populations in view of significantly increased risks. We aimed to compare the role of Epstein-Barr virus (EBV) DNA load and EBV IgA serology as tools for screening patients with a first-degree family history of NPC. STUDY DESIGN: Case-control study. SETTING: Tertiary referral center. SUBJECTS AND METHODS: Serum samples were compared from 293 newly diagnosed NPC patients and 475 individuals with a first-degree family history of NPC. EBV DNA load was measured by real-time quantitative polymerase chain reaction, while EBV viral capsid antigen (VCA) IgA and EBV early antigen (EA) IgA titers were measured by immunofluorescence assays. RESULTS: NPC patients had a significantly higher median EBV DNA load as compared with unaffected family members (835.4 vs 18.8 copies/mL; P < .001). At 100 copies/mL, EBV DNA load demonstrated a sensitivity of 76.8% and a specificity of 85.6%. A positive EBV-EA IgA titer (≥1:10) gave a sensitivity of 85.0% and a specificity of 96.4%. There was good correlation between EBV DNA load and EBV serology titers (Spearman's ρ = .536 and .594 for EBV-VCA IgA and EBV-EA IgA, respectively; P < .001). Receiver operating characteristic analysis demonstrated that EBV-VCA IgA and EBV-EA IgA were better classifiers than EBV DNA load (areas under the curve: 0.942, 0.926, and 0.880, respectively) in distinguishing NPC patients and unaffected family members. CONCLUSION: EBV DNA load and EBV IgA serology demonstrate good sensitivity and specificity as screening tools. EBV-EA IgA gave the best sensitivity and specificity profile as a screening tool for NPC among high-risk family members.

Leukocyte immunoglobulin-like receptor B1 is critical for antibody-dependent dengue.

Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub- or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies.

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1(125-133), LMP2A(426-434) or EBNA1(562-570) in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1(562-570) in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.

The structural basis for serotype-specific neutralization of dengue virus by a human antibody.

Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes. Identification of the protective determinants of the human antibody response to DENV is a vital requirement for the design and evaluation of future preventative therapies and treatments. Here, we describe the isolation of a neutralizing antibody from a DENV1-infected patient. The human antibody 14c10 (HM14c10) binds specifically to DENV1. HM14c10 neutralizes the virus principally by blocking virus attachment; at higher concentrations, a post-attachment step can also be inhibited. In vivo studies show that the HM14c10 antibody has antiviral activity at picomolar concentrations. A 7 Å resolution cryoelectron microscopy map of Fab fragments of HM14c10 in a complex with DENV1 shows targeting of a discontinuous epitope that spans the adjacent surface of envelope protein dimers. As found previously, a human antibody specific for the related West Nile virus binds to a similar quaternary structure, suggesting that this could be an immunodominant epitope. These findings provide a structural and molecular context for durable, serotype-specific immunity to DENV infection.

Differential signal transduction, membrane trafficking, and immune effector functions mediated by FcgammaRI versus FcgammaRIIa.

Receptors for the fragment crystallizable region of immunoglobulin-G (FcgammaRs) play an important role in linking the humoral and cellular arms of the immune response. In this study, we present a comprehensive functional comparison of 2 human Fc-receptors, FcgammaRI and FcgammaRIIa. Activation of FcgammaRI results in a novel signaling cascade that links phospholipase D1 to sphingosine kinase-1 in U937 cells and primary human monocytes. This induces the expression of proinflammatory mediators and is associated with trafficking of immune complexes into human leukocyte antigen-DM positive antigen-processing compartments coupled with improved MHC class II-mediated antigen presentation to T lymphocytes. In contrast, activation of FcgammaRIIa elicits signaling through phospholipase Cgamma1, resulting in increases in intracellular calcium, activation of nicotinamide adenine dinucleotide phosphate-oxidative burst, and differential membrane trafficking combined with impaired antigen presentation and proinflammatory cytokine expression. These data provide a mechanistic insight into the disparate activities associated with Fc receptors in immunity, namely, reinforcement of immune responses through stimulation of proinflammatory signaling and antigen presentation, versus the maintenance of immunologic homeostasis through the noninflammatory clearance of immune complexes.

Polysaccharide-protein complex from Lycium barbarum L. is a novel stimulus of dendritic cell immunogenicity.

Dendritic cell (DC) immunogenicity correlates with its maturation, which can be induced by toxic microbial products such as LPS. In this study, we report that a nontoxic polysaccharide-protein complex isolated from a Chinese medicinal herb, Lycium barbarum (LBP), induces phenotypic and functional maturation of DCs with strong immunogenicity. LBP up-regulated DC expression of CD40, CD80, CD86, and MHC class II molecules; down-regulated DC uptake of Ag; enhanced DC allostimulatory activity; and induced IL-12p40 and p70 production. All of its five fractions were active. LBP developed enhanced Th1 response, and LBP-treated DCs enhanced Th1 and Th2 responses in vitro and in vivo. Our study provides evidence and rationale on using LBP in various clinical conditions to enhance host immunity and suggests LBP as a potent adjuvant for the design of DC-based vaccines.

Activation of macrophages by polysaccharide-protein complex from Lycium barbarum L.

Macrophages play crucial roles in innate immunity. This paper reports that a polysaccharide-protein complex isolated from Lycium barbarum (LBP) is able to activate macrophages. LBP was isolated from Lycium barbarum fruit and separated to five homogenous fractions, designated LBPF1, LBPF2, LBPF3, LBPF4 and LBPF5. It was found that LBP (50 mg/kg, i.p.) markedly upregulated the expressions of CD40, CD80, CD86 and MHC class II molecules on peritoneal macrophages. In vitro studies showed that LBP and LBPF1-5 activated transcription factors NF-kappaB and AP-1 by RAW264.7 macrophage cells, induced TNF-alpha, IL-1beta, IL-12p40 mRNA expression, and enhanced TNF-alpha production in a dose-dependent manner. Furthermore, LBP (50 mg/kg, i.p.) significantly enhanced macrophage endocytic and phagocytic capacities in vivo. These results indicate that LBP enhances innate immunity by activating macrophages. The mechanism may be through activation of transcription factors NF-kappaB and AP-1 to induce TNF-alpha production and upregulation of MHC class II costimulatory molecules.

Activation of T lymphocytes by polysaccharide-protein complex from Lycium barbarum L.

T lymphocytes play central roles in adaptive immunity. Lycium barbarum L. (L. barbarum), also known as wolfberry, is a Chinese herbal medicine with various biological activities, such as enhancing immunity, protecting liver damage, and reducing the side effects of chemotherapy and radiotherapy. Here, we report that polysaccharide-protein complex from L. barbarum (LBP) is able to activate T cells. LBP was isolated from L. barbarum and separated to five homogenous fractions, designated LBPF1, LBPF2, LBPF3, LBPF4, and LBPF5. We found that LBP, LBPF4, and LBPF5 significantly stimulated mouse splenocyte proliferation. The proliferation proved to be of T cells, but not B cells. Cell cycle profile analysis indicated that LBP, LBPF4, and LBPF5 markedly reduced sub-G1 cells. LBP, LBPF4, and LBPF5 could activate transcription factors NFAT and AP-1, prompt CD25 expression, and induce IL-2 and IFN-gamma gene transcription and protein secretion. LBP (i.p. or p.o.) significantly induced T cell proliferation. Our results suggest that activation of T lymphocytes by LBP may contribute to one of its immuno-enhancement functions.

Asian population frequencies and haplotype distribution of killer cell immunoglobulin-like receptor (KIR) genes among Chinese, Malay, and Indian in Singapore.

Killer cell immunoglobulin-like receptors (KIR) gene frequencies have been shown to be distinctly different between populations and contribute to functional variation in the immune response. We have investigated KIR gene frequencies in 370 individuals representing three Asian populations in Singapore and report here the distribution of 14 KIR genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) with two pseudogenes (2DP1, 3DP1) among Singapore Chinese (n = 210); Singapore Malay (n = 80), and Singapore Indian (n = 80). Four framework genes (KIR3DL3, 3DP1, 2DL4, 3DL2) and a nonframework pseudogene 2DP1 were detected in all samples while KIR2DS2, 2DL2, 2DL5, and 2DS5 had the greatest significant variation across the three populations. Fifteen significant linkage patterns, consistent with associations between genes of A and B haplotypes, were observed. Eighty-four distinct KIR profiles were determined in our populations, 38 of which had not been described in other populations. KIR haplotype studies were performed using nine Singapore Chinese families comprising 34 individuals. All genotypes could be resolved into corresponding pairs of existing haplotypes with eight distinct KIR genotypes and eight different haplotypes. The haplotype A2 with frequency of 63.9% was dominant in Singapore Chinese, comparable to that reported in Korean and Chinese Han. The A haplotypes predominate in Singapore Chinese, with ratio of A to B haplotypes of approximately 3:1. Comparison with KIR frequencies in other populations showed that Singapore Chinese shared similar distributions with Chinese Han, Japanese, and Korean; Singapore Indian was found to be comparable with North Indian Hindus while Singapore Malay resembled the Thai.

Mutation screening in KCNQ1, HERG, KCNE1, KCNE2 and SCN5A genes in a long QT syndrome family.

INTRODUCTION: Long QT syndrome (LQTS), an inherited cardiac arrhythmia, is a disorder of ventricular repolarisation characterised by electrocardiographic abnormalities and the onset of torsades de pointes leading to syncope and sudden death. Genetic polymorphisms in 5 well-characterised cardiac ion channel genes have been identified to be responsible for the disorder. The aim of this study is to identify disease-causing mutations in these candidate genes in a LQTS family. MATERIALS AND METHODS: The present study systematically screens the coding region of the LQTS-associated genes (KCNQ1, HERG, KCNE1, KCNE2 and SCN5A) for mutations using DNA sequencing analysis. RESULTS: The mutational analysis revealed 7 synonymous and 2 non-synonymous polymorphisms in the 5 ion channel genes screened. CONCLUSION: We did not identify any clear identifiable genetic marker causative of LQTS, suggesting the existence of LQTS-associated genes awaiting discovery.

A population-based LD map of the human chromosome 6p.

The recent publication of the complete sequence of human chromosome 6 provides a platform from which to investigate genomic sequence variation. We report here a detailed linkage disequilibrium (LD) pattern map across the entire human chromosome 6p by using a set of 1152 single nucleotide polymorphisms (SNPs) in a population of 198 Singaporean Chinese, with 326 SNPs focused in the major histocompatibility complex (MHC) region. Our analysis shows some unexpectedly high segments of strong LD in a 10-Mb region that includes the extremely polymorphic and gene-rich MHC loci and many non-MHC genes. These include the telomeric peri-MHC region that harbors olfactory receptors, histones and zinc finger clusters, and the centromeric peri-MHC region that contains several unknown open reading frames. The data also help refine a human-mouse synteny break in the region between 28.6 and 29.4 Mb. The population-based LD map presented here will provide an essential resource for understanding the genomic sequence variation of chromosome 6p and LD mapping of disease genes of complex genetic traits.

Host heterogeneous ribonucleoprotein K (hnRNP K) as a potential target to suppress hepatitis B virus replication.

BACKGROUND: Hepatitis B virus (HBV) infection results in complications such as cirrhosis and hepatocellular carcinoma. Suppressing viral replication in chronic HBV carriers is an effective approach to controlling disease progression. Although antiviral compounds are available, we aimed to identify host factors that have a significant effect on viral replication efficiency. METHODS AND FINDINGS: We studied a group of hepatitis B carriers by associating serum viral load with their respective HBV genomes, and observed a significant association between high patient serum viral load with a natural sequence variant within the HBV enhancer II (Enh II) regulatory region at position 1752. Using a viral fragment as an affinity binding probe, we isolated a host DNA-binding protein belonging to the class of heterogeneous nuclear ribonucleoproteins--hnRNP K--that binds to and modulates the replicative efficiency of HBV. In cell transfection studies, overexpression of hnRNP K augmented HBV replication, while gene silencing of endogenous hnRNP K carried out by small interfering RNAs resulted in a significant reduction of HBV viral load. CONCLUSION: The evidence presented in this study describes a wider role for hnRNP K beyond maintenance of host cellular functions and may represent a novel target for pharmacologic intervention of HBV replication.

Seroepidemiology of neutralizing antibodies to Japanese encephalitis virus in Singapore: continued transmission despite abolishment of pig farming?

Japanese encephalitis virus (JEV) transmission in Singapore appeared to have ceased after pig farming in Singapore was phased out from the early 1980s. However, the recent detection of neutralizing antibodies to JEV in a population of wild boars in an offshore island, as well as the notification of two human cases of JE in Singapore in 2001, prompted us to reconsider the presence and hence the public health threat of JEV in Singapore. We report here a serological study of animals, birds and humans for neutralizing antibodies to JEV. The results indicate that JEV may still be actively transmitted in the peripheral part of the Singapore island and that regular serological surveys of farm animals and birds, such as chickens, may be useful to further elucidate the activity of JEV in Singapore.

Induction of acute graft-versus-host disease by T cells that do not respond to in vitro alloantigen stimulation.

The mixed lymphocyte reaction (MLR) has been used extensively to measure alloreactive T cells. In clinical practice, a negative MLR of recipient T cells responding to donor cells does not necessarily mean that a donor-specific tolerance has been established. This discrepancy indicates that the presently used methods fail to demonstrate the full repertoire of alloreactive T cells. This could be the result of the fact that some alloreactive T cells do not respond in vitro but will mount a response towards alloantigens in vivo, or that some alloreactive T cells do not respond during the MLR but will respond later if the alloantigen stimulation remains. We therefore examined the non-proliferating T-cell population in a mouse primary alloreactive response. Spleen and lymph node cells derived from C57BL/6J (H-2(b)) mice were stained with carboxy-fluorescein diacetate succinimidyl ester and injected intravenously into C.B-17 severe combined immunodeficient (SCID) mice (H-2(d)). The donor cells were recovered 5 d after the injection. The non-proliferating T cells were sorted and were non-reactive to alloantigen stimulation in vitro. Nevertheless, these non-proliferating T cells could proliferate and cause acute graft-versus-host disease after being adoptively transferred to secondary recipients of SCID mice. These results suggest that there exists an alloreactive T-cell population that does not respond to in vitro alloantigen stimulation but can mount a delayed alloresponse in vivo.

Expression of interleukin-18 by nasopharyngeal carcinoma cells: a factor that possibly initiates the massive leukocyte infiltration.

Interleukin-18 (IL-18) is a single-chain cytokine that is produced by various cells. With interleukin-12 (IL-12), it synergistically stimulates activated T cells and natural killer (NK) cells to produce interferon-gamma (IFN-gamma). Nasopharyngeal carcinoma (NPC) is the most common form of nasal and nasopharyngeal malignancy, and in NPC tumor tissues there is an intense leukocyte infiltration comprising predominantly T cells and macrophages. We previously showed an increased expression of IFN-gamma in the infiltrating T cells. To identify the cells that provide IL-12 and IL-18 for stimulating the expression of IFN-gamma in activated T cells, NPC cell lines CNE-2 and HK-1, as well as biopsies obtained from NPC and control individuals, were examined. CNE-2 and HK-1 cells were found to express messenger RNA encoding IL-18, but not IL-12. Secreted IL-18 was detected in the culture supernatant. Addition of a caspase-1 inhibitor decreased the secretion level, indicating that this IL-18 secretion was caspase-1 dependent. Moreover, the in vitro IL-18 production in NPC cell lines correlated with the NPC tumor cells in situ. NPC tumor cells in the biopsies produced IL-18, as detected by immunohistochemistry and immunofluorescent double staining. In contrast, IL-18 expression was not observed in the control biopsies. We suggest that IL-18 secreted by NPC tumor cells plays a role in initiating the leukocyte infiltration process. IL-18 stimulates T cells and NK cells to produce IFN-gamma, which consequently activates macrophages and other immune cells to secrete chemokines to start a leukocyte recruitment cascade.