Search

We aimed to evaluate the effectiveness of nasopharyngeal cancer (NPC) screening by comprehensive clinical follow-up and adjunctive Epstein-Barr virus (EBV) testing. In a prospective cohort study, 524 individuals with a first-degree family history of NPC were recruited at a university clinical center in Singapore. The cohort was evaluated at baseline and at 6 monthly intervals, with a complete head and neck examination including nasopharyngeal endoscopy. Blood was taken at baseline and at yearly intervals for EBV Viral Capsid Antigen (VCA) IgA, EBV Early Antigen (EA) IgA serology and serum cell-free EBV DNA. Nasopharyngeal biopsy was performed when any irregularity in the nasopharynx was observed, or when EBV markers were elevated. The mean duration of follow-up was 57.7 months, with an average of 8.6 clinical visits per participant. Five participants (0.96%) were identified to have NPC, giving a prevalence of 199 per 100,000 person-years of screening. Four of the five NPC cases identified had asymptomatic T1 disease, at an earlier stage compared to NPC patients diagnosed in the clinic during the same time period (p = 0.0297). All NPC cases identified had elevated EBV-EA IgA titers ≥1:10, with a specificity of 94.6% and a positive predictive value of 15.2%, outperforming EBV-VCA IgA and serum EBV DNA. Two NPC cases were biopsied only because of elevated EBV serology titers, with increasing EBV-EA IgA titers preceding the diagnosis of NPC. In conclusion, screening for NPC is effective in identifying early-stage disease. Adjunctive EBV-EA IgA testing improved the effectiveness of screening.

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.

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.

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.Case-control study.Tertiary referral center.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.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.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.

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.

Receptors for the fragment crystallizable region of immunoglobulin-G (FcγRs) 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, FcγRI and FcγRIIa. Activation of FcγRI 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 FcγRIIa elicits signaling through phospholipase Cγ1, 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.

The HLA profiles of three common populations from South East Asia, Chinese and Malays from Singapore and Filipinos from the Philippines are documented.

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.

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.

The direct involvement of the human leukocyte antigen class II DR-DQ genes in type 1 diabetes (T1D) is well established, and these genes display a complex hierarchy of risk effects at the genotype and haplotype levels. We investigated, using data from 38 studies, whether the DR-DQ haplotypes and genotypes show the same relative predispositional effects across populations and ethnic groups. Significant differences in risk within a population were considered, as well as comparisons across populations using the patient/control (P/C) ratio. Within a population, the ratio of the P/C ratios for two different genotypes or haplotypes is a function only of the absolute penetrance values, allowing ranking of risk effects. Categories of consistent predisposing, intermediate ('neutral'), and protective haplotypes were identified and found to correlate with disease prevalence and the marked ethnic differences in DRB1-DQB1 frequencies. Specific effects were identified, for example for predisposing haplotypes, there was a statistically significant and consistent hierarchy for DR4 DQB1*0302s: DRB1*0405 =*0401 =*0402 > *0404 > *0403, with DRB1*0301 DQB1*0200 (DR3) being significantly less predisposing than DRB1*0402 and more than DRB1*0404. The predisposing DRB1*0401 DQB1*0302 haplotype was relatively increased compared with the protective haplotype DRB1*0401 DQB1*0301 in heterozygotes with DR3 compared with heterozygotes with DRB1*0101 DQB1*0501 (DR1). Our results show that meta-analyses and use of the P/C ratio and rankings thereof can be valuable in determining T1D risk factors at the haplotype and amino acid residue levels.

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 wellcharacterised 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 nonsynonymous 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 LQTSassociated genes awaiting discovery. Key words: Arrhythmia, Ion channels, Long QT syndrome

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.

Summary 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-2b) mice were stained with carboxy-fluorescein diacetate succinimidyl ester and injected intravenously into C.B-17 severe combined immunodeficient (SCID) mice (H-2d). 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.

Rheumatoid arthritis in Singaporean Chinese has previously been shown to be associated with the DRB1*0405, DRB1*1001 haplotypes and to the DRB1*0901 haplotype when the former two were removed. The present paper focused on eight HLA associated microsatellite markers (TNFa, TNFd, D6S273, TAP1CA, DQCAR, DQCARII, D6S2222, D6S2223) and their allelic associations with Chinese RA. 60 RA patients and 75 healthy controls were studied. It appeared that DQCARII*194/DRB1*0405/TNFa*117 was part of the extended haplotype predisposed to RA, whereas DRB1*0901/D6S273*128 contributed to susceptibility to RA to a lesser degree in Singaporean Chinese. Additionally, a negative association with DQCAR*186/DRB1*0301/D6S273*122/TNFd*124 was observed. No association with disease development was observed in this study.

Acute graft-versus-host disease (GVHD) involves mainly skin, liver and intestines. Other organs such as heart, muscle and central nervous system are seldom affected, although their parenchymal cells also express alloantigens, such as MHC class I antigens. The mechanism of this selective involvement of distinct organs in acute GVHD is not well understood. We postulated that it might be related to the selective migration of activated alloreactive T cells. Indeed, T cell infiltration, revealed by examination of serial samples using flow cytometry and immunohistology, occurred early and continuously in the target organs such as the liver, but not in a non-target organ, the heart, in a murine acute GVHD model. Since T cell migration is largely controlled by the expression of chemokine and chemokine receptors, we investigated the chemokine spectrum in target/non-target organs of mice with acute GVHD. We found that in the spleen and liver MIP-1alpha, MIP-2 and Mig were the predominant chemokines expressed. In another target organ, the skin, MIP-1alpha, MIP-2, MCP-1 and MCP-3 were all highly expressed. In a non-target organ of acute GVHD, the heart, the predominant chemokines expressed were MCP-1 and MCP-3. This distinct pattern of chemokine expression in these organs may contribute to the preferential recruitment of inflammatory cells into the liver and skin, but not into the heart, in acute GVHD.

Human leukocyte antigen (HLA) associations have been frequently reported in childhood steroid-responsive nephrotic syndrome (SRNS) in other populations. The aim of this study was to characterize the immunogenetic background of Singaporean Chinese patients with childhood SRNS. We determined the HLA class I (HLA- A* and HLA-B*) as well as class II (HLA- DRB1*, HLA- DQB1*) gene polymorphisms using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique, in patients with SRNS (n=64) and normal controls (n=236 for HLA- A*, n=80 for HLA- B*, HLA- DRB1* and HLA- DQB1*). The frequency of HLA- A*11 allele was significantly higher in the SRNS patients compared to controls (78.1% vs 54.2%, respectively; relative risk, RR=3.01, Pc=0.011). However, there was no significant difference in the allele frequencies of HLA- B*, HLA- DRB1* and HLA- DQB1* between the SRNS patients and controls, unlike that in previous studies. Our data suggest that the immunogenetic background of Singaporean Chinese with childhood SRNS was different from that in other populations. As HLA- A*11 has been strongly associated with other autoimmune diseases, it is conceivable that the HLA- A*11-specific motif may play a role in the development of the abnormal T-cell-mediated immune response that may be responsible for triggering the proteinuria seen in SRNS.

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.

L-selectin, LFA-1 and alpha(4) integrins play important roles in the homing of naive T cells into peripheral lymphoid tissues. L-selectin- or LFA-1-deficient lymphocytes cannot effectively home to lymph nodes (LN), and antibody blockade of alpha(4) integrins also hinders lymphocytes homing. The present study was initiated to explore whether it is feasible to ameliorate acute graft-versus-host disease (aGVHD) by modulating the homing process of donor cells in the recipient in a mouse model. Using a fluorescence labeling method, we found that two monoclonal antibodies directed at L-selectin and alpha(4) integrins, respectively, when used in combination, could delay half of the donor C57BL/6J mouse spleen cells homing into the LN of recipient BALB/c mouse 15 h after injection. Spleen cells (1 x 10(7)) derived from C57BL/6J (H-2(b)) mice were injected into each C.B-17 SCID recipient mouse (H-2(d)) with or without prior incubation with 10 microg each of the two antibodies. T cell repopulation in the blood was observed in both groups of mice at a comparable level 14 days after injection of the donor cells. Eight control mice started to show aGVHD signs 7 - 14 days after the injection, and all died by day 31. However, among the ten mice that received the antibody-treated donor cells, two died before day 29, four survived between 36 and 78 days, and the remaining four survived more than 150 days, with two of them aGVHD free. It is apparent that the temporarily reduced lymphocyte homing into LN reduced the alloreactivity of the donor T cells, thus providing a simple way of modifying aGVHD. This novel approach may shed light on the prevention of aGVHD associated with clinical bone marrow transplantation.

Nasopharyngeal carcinoma (NPC) is characterized by harboring Epstein-Barr virus genes in the tumor cells and an intense infiltration of leukocytes in the tumor tissue. These infiltrating cells are mainly composed of T lymphocytes and macrophages. The mechanism of this intense infiltration has long been a puzzle. We attempted to address this issue by studying the expression of CC chemokines, which are responsible for recruiting both T cells and macrophages, by an immunohistochemical approach. In biopsies obtained from nasopharynx of 17 NPC patients that contained tumor cells, expression of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, macrophage chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, and RANTES was detected in the tumor-infiltrating cells, with MIP-1alpha and MCP-1 found in nearly all biopsies and the others relatively less frequently. Furthermore, expression of interferon-gamma (IFN-gamma) was also observed in tumor-infiltrating cells. In contrast, CC chemokines and IFN-gamma were rarely expressed in the 13 control biopsies that were either normal or with nonspecific inflammation, and in 4 biopsies from untreated NPC patients that contained no tumor cells. Using an immunofluorescent double-staining method, MIP-1alpha and MCP-1 were identified to be associated with macrophages, and IFN-gamma with T cells. Moreover, expression of CCR2 and CCR5, the receptors for these chemokines, was also detected in the tumor-infiltrating cells. These data indicate that the intense tumor infiltration by T cells and macrophages is a result of active recruitment. It seems possible that the intense infiltration of leukocytes in NPC tumor tissue is initiated by the activated tumor-reactive T cells. T cells migrate into the tumor tissue in an antigen-specific mode, and IFN-gamma secreted from these pioneer T cells activates tissue macrophages to express CC chemokines, especially MIP-1alpha and MCP-1, which consequently recruit more T cells and macrophages into the tumor tissue.

Tumour cells of undifferentiated nasopharyngeal carcinoma (NPC) consistently harbour Epstein–Barr virus (EBV) genes. Expression of mRNA transcripts associated with EBV latency has been demonstrated in such cells. However, expression of EBV lytic genes has not been well elucidated, although various lines of evidence have suggested that there is EBV replication in NPC tumour cells. We have studied mRNA expression of representative EBV lytic genes by RT–PCR in nasopharynx biopsies obtained from NPC and control individuals. In both NPC and control biopsies, EBV lytic genes BZLF1, BALF2 and BCLF1 were detected readily. However, BRLF1 was detected in NPC biopsies only. The BRLF1 gene was then cloned and expressed in vitro, and the protein product, Rta, was used as an antigen to detect specific antibodies by immunoprecipitation in plasma samples obtained from NPC patients and healthy controls. IgG antibodies directed against Rta were detected in 44 of 53 NPC plasma samples (83·0%), but only in 1 of 53 control samples (1·9%). Furthermore, the antibody binding regions were found in the C-terminal two-thirds of Rta. This serological result confirms indirectly that BRLF1 is specifically expressed in NPC tumour cells. Rta might play an important role in NPC pathogenesis, considering its multiple functions in EBV replication and cell cycles. Moreover, the detection of IgG antibodies directed against Rta could be developed into a diagnostic parameter for NPC.

In an attempt to clone and express proteins from the Epstein-Barr virus (EBV) cDNA library to be used as antigens in an enzyme-linked immunosorbent assay (ELISA) format to test against the antibodies found in the sera of nasopharyngeal carcinoma (NPC) patients, we have isolated and characterized three clones. All three clones expressed the same polypeptides of different lengths, which belong to the carboxyl terminal end of the large subunit of ribonucleotide reductase (RR) of the EBV genome. All three clones were found to be immunogenic and could be used in an IgA and IgG ELISA against the NPC sera with various degrees of sensitivity and specificity. Because the clones varied in length, this difference provides a simple system to determine where most of the antibody epitopes lies on the protein. We designed an immunoabsorption assay and a mathematical model to help map the segment of the polypeptide most immunogenic to 43 NPC patients. Results were unexpected: 77% of the patients were most immunogenic to region z, which was the smallest fragment among the three fragments studied. Fragment z was only 33 amino acids in length. Only 14% and 19% of patients showed the most immunogenic region in segment x and y, respectively. This variation could be due to major histocompatibility complex antigens. The patients could be divided into three groups based on the immunoabsorption assays, in which each group responded to a different immunodominant segment in the RR antigen. The largest group responded to an immunodominant segment, which was only 33 amino acids long. This domain was coded for by the gene fragment from nucleotide 78,129 to nucleotide 78,227 of the EBV genome. This segment of the protein would be suitable for further epitope mapping studies.

SUMMARY Increased serum levels of interferon-c (IFN-c) have been observed in acute graft-versus-host disease (GVHD). Recent in vitro studies have demonstrated that interleukin-12 (IL-12) and interleukin-18 (IL-18) synergistically up-regulate IFN-c secretion. In this communication, we investigated the factors relevant to IFN-c secretion in acute GVHD. A murine model of acute GVHD was established by injecting donor spleen cells into severe combined immunodeficiency (SCID) mice. A series of specimens, including sera, livers and spleens derived from the GVHD mice, were investigated with histological examination, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and semiquantitative reverse transcription‐polymerase chain reaction (RT‐PCR). IFNc secretion increased in serum 3 days after spleen cell transfer, peaked on day 7, and then gradually decreased close to the baseline level by day 35. A synchronized increase of activated T cells and mRNA expression of IL-12, IL-18 and their respective receptors was observed after spleen cell transfer. However, only the kinetic expression pattern of IL-12 receptor (IL-12R) b2 chains was closely correlated with that of IFN-c, while IL-12 dropped to the baseline level earlier than IFN-c. Therefore, IFN-c expression in the early phase of acute GVHD is a mono-peak and self-restricted pattern. Its secretion is closely related with T-cell activation, the presence of IL-12, IL-18 and their respective receptors. However, the limiting factors for IFN-c secretion seem to be IL-12 and IL-12R b2 chains.

The occurrence of GBV-C/HGV infection was studied in 160 individuals from two high-risk groups in Singapore. RT-PCR of the 5'-UTR detected GBV-C/HGV RNA in 3/73 (4.1%) of patients undergoing maintenance hemodialysis for chronic renal failure, and in 17/87 (19.5%) of patients coinfected with HCV who tested positive for HCV RNA. Phylogenetic analysis of 5'-UTR sequences from these 20 samples showed that the Asian or group 3 variant was found in 45% of the samples sequenced, thus confirming the high frequency of this variant in the region. Group 2a variants accounted for 50% of the samples with a complete absence of group 2b. Our analysis also provided strong bootstrap support for the subdivision of group 2 into subgroups 2a and 2b. This study shows that isolates belonging to all three main groups of GBV-C/ HGV can be detected in Singapore, with the large majority belonging to groups 2a (50%) and 3 (45%). Only a single group 1-like sequence was detected within the 20 isolates. Of interest also is that all group 3 isolates were identified in Chinese patients while group 2a was found in both Chinese and Malay.

A pair of enantiomeric gold(I) complexes containing enantiomerically pure 2,3-dimethyl-7-phenyl-5-(phenylsulfanyl)-7-( R/S )-phosphabicyclo[2.2.1]hept-2-ene has been prepared and in vitro cytotoxicity tests showed that both the enantiomers are relatively nontoxic against healthy lymphocytes, but are highly potent against suspension and monolayer human tumor cell lines.

The organopalladium complex containing orthometalated (S)-(1-(dimethylamino)ethyl)naphthalene as the chiral auxiliary has been used successfully to promote the asymmetric [4+2] Diels−Alder reaction between 3,4-dimethyl-1-phenylphosphole and 2-methylene-3-quinuclidinone. In this reaction, the organopalladium template exhibited remarkable stereochemical and electronic directing effects such that the quinuclidinone-nitrogen atom in the resulting phosphanorbornene cycloadduct is located stereospecifically in the exo position. Only one enantiomerically pure P−N bidentate ligand was obtained, although four diastereomeric products are possible. The absolute configuration and the coordination properties of the P-chiral cycloadduct have been established by single-crystal X-ray analyses.

Nasopharyngeal carcinoma (NPC) has been known to be associated with specific HLA haplotypes, in particular HLA A2, B46 and A33, B58 haplotypes. A linkage study based on this observation suggested that HLA antigens are not the cause of NPC but that there exists a gene that lies close to if not within the major histocompatibility complex locus and confers a greatly increased relative risk of NPC. Since then, no further work has elucidated the presence of this gene. One of the difficulties faced by researchers has been the size of the region of chromosome implicated. The MHC locus alone is almost 4 Mb in length, and the number of genes encoded within it is numerous. The purpose of our study was thus to reduce the region of DNA in which the NPC susceptibility gene is likely to be. We report that the NPC susceptibility gene may be within the centromeric end of the class-1 and the telomeric end of the class-III regions of the MHC, near the D6S1624 microsatellite locus, where the presence of allele 4 of the microsatellite conferred a 3 1/2-fold increase in the risk of NPC, the highest reported for a single locus, and the presence of allele 1 of the same microsatellite conferred a highly significant protective effect against NPC.

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 LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 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.

Introduction: Epstein-Barr virus (EBV) is a gamma herpesvirus that is found in the majority of the human population. Though commonly established as a life-long, asymptomatic infection, EBV has also been implicated in a number of human malignancies including Hodgkin's and Burkitt's lymphomas, nasopharyngeal carcinoma as well as post-transplant lymphoproliferative disease in immunosuppressed patients. While EBV displays restricted gene expression during different latency programs, the viral gene products that are frequently detected in EBV-associated tumors include Epstein-Barr virus Nuclear Antigen 1 (EBNA1), Latent Membrane Protein 1 (LMP1) and Latent Membrane Protein 2A (LMP2A). Our lab has previously described the generation and characterization of monoclonal antibodies with T cell receptor-like specificities (TCR-like mAbs) targeting EBV latent epitopes in association with HLA-A0201. These antibodies have been shown to bind to their targets with high specificities and affinities, and were capable of recognizing antigens displayed on human nasopharyngeal carcinoma biopsies, highlighting their immunotherapeutic potential for targeting EBV-associated tumors. Methods: EBV is particularly capable of transforming resting B cells into latently infected, actively proliferating B lymphoblastoid cell lines (BLCL) in vitro. As a proof-of-concept, we first generated EBV BLCLs from HLA-A0201+ human peripheral blood mononuclear cells (PBMCs) and compared the ability of our TCR-like mAbs to detect their endogenous targets before and after establishment of the cell lines. The xenograft model involving the inoculation of EBV BLCL into immunodeficient mice resembles B cell lymphoma that develops in post-transplant, immunosuppressed patients. Hence to test the ability of our TCR-like mAbs to target the BLCLs in vivo, we next inoculated PBMC-derived BLCLs into immunodeficient NSG mice and administered weekly dosage of TCR-like mAbs. Mice were assessed for changes in weight, survival as well as end-point organs analysis. Results: Here, we showed that endogenous EBV latent epitopes could be detected by our TCR-like mAbs on PBMC-derived BLCLs after EBV establishment of the cell line. Despite the inherent heterogeneities between different donors PBMC-derived BLCLs, several of the HLA-A0201+ BLCLs tested displayed similar binding activities with these TCR-like mAbs. In addition, BLCL-injected NSG mice that received weekly treatment of TCR-like mAbs displayed reduced tumor burden, splenomegaly and liver tumor spots in comparison to mice that received isotype antibody or PBS control. Importantly, BLCL-injected NSG mice that received the TCR-like mAb targeting the EBNA1 latent epitope exhibited delayed weight loss and survival advantage. Conclusion: We have utilized TCR-like mAbs with specificities against EBV latent epitopes expressed on HLA-A0201 and showed that these antibodies could recognize endogenous targets on EBV transformed, PBMC-derived BLCLs. Furthermore, weekly administration of TCR-like mAbs into BLCL xenograft NSG mice resulted in an overall reduction of tumor load and improved survival in one of the three described antibodies. Taken together, our data provide preliminary evidence for the therapeutic usage of antibodies with TCR-like specificities for the targeting EBV transformed BLCL in vivo. Citation Format: Junyun Lai, Wei Jian Tan, Chien Tei Too, Nalini Srinivasan, Lan Hiong Wong, Fatimah Mustafa, Soh Ha Chan, Jianzhu Chen, Paul Anthony MacAry. Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B095.

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.

This study analysed HLA class 1 and 2 allele associations in Singaporean Chinese patients with RA. Seventy patients (ARA definite or classical) and 80 controls were typed for HLA class 1 alleles by serology and class 2 alleles by serology and the PCR/SSO method. RA patients had higher frequencies of DRB1*0405 (40 vs 12.5%; corrected probability value (PC) < 0.02, relative risk (RR) = 4.7, 95% confidence limit (CL) 2.1-10.6), DRB1*1001 (14.3 vs 1.3%; PC = 0.06, RR = 13.2, 95% CL 1.6-105.7), DQB1*0401 (38.6 vs 12.5%; P = 0.006, RR = 4.4, 95% CL 1.9-10.0) and DQB1*0501 (20 vs 5%; PC = 0.048, RR = 4.8, 95% CL 1.5-15.2). It is concluded that Chinese RA is associated primarily with HLA DRB1*0405 and DRB1*1001 which share common amino acid sequences in the third hypervariable region of the DR beta chains shown to be associated with RA in other ethnic groups. Patients without DRB1*0405 and *1001 had a higher frequency of DRB1*0901, which is in linkage disequilibrium with HLA B46 in the Chinese.

A monoclonal antibody 137BL7 raised against purified DR1 protein was shown to bind specifically to 5/5 DR1, 18/18 DR2 cells and 0/23 non-DR1,2 cells by cell-EIA. Further analysis by FACS using HLA-transfectants revealed that 137BL7 also bound specifically to the DRB5 transfectants in addition to the expected DR1 transfectants. However, it did not bind to the DR2 (DR15) transfectants, showing that cross-reactivity with DR2 cells lies with the DRB5 (DR51) rather than the DRB1 gene product. A comparison of the HLA-DR amino acid sequences of DR1 and DR51 antigens revealed a common glutamic acid residue at position 96, which may form the putative binding epitope of this mAb.

Chinese Singaporean myasthenia gravis (MG) patients are associated with three HLA haplotypes: Cw1 B46 DRB1*0901 DQB1*0303 DQA1*03, DRB1*14 DRB3*0202 DQB1*0503 DQA1*0101 and DRB1 *1202 DRB3*0301 DQB1*0301 DQA1*0601. The B46 haplotype was associated with the total group of MG patients but in particular with those with younger onset, low anti-acetylcholine receptor (anti-AchR) titres, with only ocular lesions and with normal thymuses. The DRB1*14 haplotype was associated with thymic hyperplasia, younger onset patients, with high anti-AchR titres and with generalised MG. The DRB1 *1202 haplotype was associated with thymoma, older onset patients, ocular lesions, and mid to high anti-AchR titres.

HLA-C*03:85 differs from C*03:03:01 by a single nucleotide substitution at position 276, in exon 2.

Liver specificity of hepatitis B virus (HBV) replication has been attributed to the action of its second enhancer (EII). We report here the characterization of EII and the subsequent isolation of a novel liver-specific DNA-binding protein which binds to and activates EII. The cDNA clone of the protein, designated E2BP, was isolated from a lambda gt11 expression library constructed from the hepatoma cell line HuH-6 which was screened with a binding site probe derived from EII. Sequence analysis of E2BP revealed 86.6% homology with a rat heterogeneous nuclear ribonucleoprotein C protein sequence, while conformational studies suggest a helix-loop-helix motif as a DNA-binding site. Cloned E2BP expressed in human fibroblasts by transient transfection displayed EII binding and activating characteristics similar to those of native E2BP in hepatocytes.

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.

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.

A novel allele, C*0406 1, has been identified and is characterised by a single nucleotide substitution at position 196 of exon 3 when compared with its closest related allele, C*0403. The latter is found in 4/69 Chinese and 7/80 Malays while Cw*0406 was found in only one Malay individual within the study populations. The data suggest that Cw*0406 may have arisen as a relatively recent genetic event either by gene conversion or as a simple point mutation variant of Cw*0403.

Background Nasopharyngeal carcinoma (NPC) arises from the epithelial tissue of the nasopharynx. This cancer has remarkable ethnic and geographic distributions, with a particularly high prevalence in Southern China and Southeast Asia. Genetic susceptibility and Epstein-Barr virus infections have been implicated in the pathogenesis of NPC. However, genomic abnormalities of this neoplasm remain largely obscure, and no effective targeted therapy has been established. Therefore, a strong need exists to characterize the genetic alterations of this type of tumour for guiding the development of more effective and innovative therapeutic regimens. Methods Whole exomes sequencing (WES) was performed on 56 NPC germline/tumor pairs and 5 NPC cell lines. Transcriptome sequencing (RNA-seq) was conducted on 4 tumors from this cohort. An additional 61 NPC germline/tumor pairs and 5 non-paired primary tumors were further subjected to targeted regional deep-sequencing (TS). The somatic copy number variations (SCNV) of 52 primary tumors were profiled by SNP-array hybridization (50 of them also had WES data). Finally, both in vitro and in vivo biological and biochemical experiments were performed to evaluate the newly-identified mutations in NPC cell lines. Results We identified a total of 1,577 non-silent somatic mutations affecting 1,413 genes from WES, revealing a relatively low mutational rate and wide mutational diversity. Integration of the results from WES, TS and RNA-seq revealed a distinct mutational signature and a number of significantly mutated genes (such as TP53, ARID1A, MLL2, BAP1, PIK3CA, etc.) in NPC. Pathway enrichment analysis of genetic lesions identified several important cellular processes and pathways in NPC including chromatin modification, ERBB-PI3K signaling and autophagy machinery. We further characterized the biological functions of both ARID1A and BAP1 proteins in NPC cell lines. Depletion of wild-type endogenous ARID1A expression with shRNAs resulted in increased anchorage-independent colony formation, cell migration and xenograft growth in vivo, whereas ectopically expressed ARID1A suppressed both cell proliferation and migration. Similarly, ectopic expression of wild-type BAP1 suppressed anchorage-independent colony formation of NPC cells. Conclusion We characterized the genomic landscape of 128 NPC cases, and identified a number of novel driver genes with statistical and biological evidence. Integrated analysis showed enrichment of genetic lesions affecting chromatin modification, ERBB-PI3K signaling and autophagy machinery, offering opportunities for developing novel treatments. These results in aggregate provide an important genetic foundation for further study of the molecular pathology and etiology of this fatal disease. Citation Format: DECHEN LIN, Xuan Meng, Masaharu Hazawa, Yasunobu Nagata, Ana Maria Varela, Liang Xu, Yusuke Sato, Li-Zhen Liu, Ling-Wen Ding, Arjun Sharma, Boon Cher Goh, Soo Chin Lee, Bengt Fredrik Petersson, Feng Gang Yu, Paul Macary, Min Zin Oo, Soh Ha Chan, Henry Yang, Seishi Ogawa, H. Phillip Koeffler. The genomic landscape of nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3939. doi:10.1158/1538-7445.AM2015-3939

A chiral organopalladium complex promoted asymmetric Diels−Alder reaction between 1-phenyl-3,4-dimethylphosphole and [(E)-2-(ethoxycarbonyl)vinyl]diphenylphosphine gives a novel optically pure P-chiral diphosphine which bears an ester functional group on the carbon skeleton.

HLA Class I-restricted CD8(+) T-cell responses are believed to play an important role in controlling Epstein-Barr virus (EBV) infection, which has been consistently associated with nasopharyngeal carcinoma (NPC). Immediate early transactivator Rta of EBV has been shown to be associated with the reactivation of EBV from latency and drive the lytic cascade of EBV and comprise an important target for EBV-specific cellular cytotoxicity. Furthermore, BRLF1 is specifically expressed in NPC tumor cells. The protein product of BRLF1, Rta, could then be considered as a NPC tumor antigen. Therefore, cellular immunity against Rta represents a very important part of the immunity against NPC, as they should prevent the replication of EBV. In the present study, Rta-specific CD8(+) T-cell responses in healthy virus carriers were characterized by using A1101 tetramer containing the known Rta epitope ATIGTAMYK (134-142). We clearly showed A1101/ATIGTAMYK tetramer-reactive CD8(+) T cells in the circulation of healthy virus carriers, ranging from 2.13 to 9.03%. We then studied the expression of perforin and interferon-gamma (IFN-gamma) secretion in these Rta-specific T cells. Our study demonstrated that Rta-specific T cells are capable of IFN-gamma production and nearly 90% of the Rta-specific CD8(+) T cells expressed perforin. Presumably, these are the cells that play an important role in determining the initiation of the lytic cycle or the clearance of EBV.

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.

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., A host protein -hnRNP K- may have a crucial role in determining how well the Hepatitis B virus replicates in humans.

Nasopharyngeal carcinoma (NPC) is a human epithelial tumor with a high incidence in Southern Chinese population, with contributions from Epstein-Barr virus (EBV), human leukocyte antigen (HLA), and environmental factors to its etiology. It has been shown previously that the recognition of immediate-early transactivator Rta of EBV by CD8+ T cells may have a significant impact on controlling EBV and, indirectly, NPC. The current study used two computer-aided prediction methods and competition-based HLA-peptide binding assays to screen for HLA B2704/B4601/B5801 restricted T-cell epitopes derived from Rta. HLA tetrameric complexes containing these potential T-cell epitopes were synthesized. Rta-specific CD8+ T-cell responses in healthy virus carriers were then defined by these tetramers and IFN-gamma ELISPOT assays. We clearly demonstrated that healthy virus carriers have detectable Rta-specific CD8+ T cells restricted by B2704 in the circulation. However, there were no B4601/B5801 tetramer-reactive T cells specific for Rta in the peripheral blood of matched/mismatched donors. On the other hand, B4601 tetramers containing the computer-predicted B4601 binder EBNA3A (318-326) showed detectable tetramer-reactive T cells in the circulation of healthy virus carriers. topes also elicited IFN-gamma responses as detected by ELISPOT.

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.