| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,
* Graduate Program in Immunology/Virology,
Program in Molecular Medicine, and
Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA 01605
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A marked expansion of T lymphocytes and high frequencies of EBV-specific CD8+ T cells have been documented during AIM. Although lytic and latent viral proteins are likely expressed during primary EBV infection, early EBV-specific CD8+ T cell responses during acute infection are directed toward lytic proteins (3, 4, 5). After resolution of the acute infection, lytic epitope-specific CD8+ T cells decline in frequency, but most remain detectable throughout latency. However, latent epitope-specific CD8+ T cells are not commonly detected in the peripheral blood at presentation with AIM (4) but become detectable after several weeks. Frequencies of latent epitope-specific CD8+ T cells are generally lower than frequencies of lytic epitope-specific CD8+ T cells during AIM and remain stable over long periods of time.
Although EBV-specific CD8+ T cell responses have been well documented, EBV-specific CD4+ T cell responses have been less well studied, especially during AIM. Several groups have derived EBV-specific CD4+ T cell lines from latently infected individuals and have used these lines to determine the EBV protein and epitope specificity of CD4+ T cell responses (6, 7, 8, 9). For example, Epstein-Barr nuclear Ag (EBNA)-1- and EBNA-3C-specific CD4+ T cell lines have been generated from healthy, EBV-seropositive individuals (6, 7). In addition, Leen et al. (10) have identified HLA class II-restricted EBNA-1, EBNA-3C, latent membrane protein-1, and latent membrane protein-2 epitopes using ELISPOT assays.
Little is known about the timing, magnitude, breadth, or specificity of CD4+ T cell responses during acute EBV infection. White et al. (9) have reported the isolation of a BHRF-1-specific CD4+ CTL clone from the peripheral blood of an AIM patient, but more direct characterization of EBV-specific CD4+ T cell responses and their relationship to CD8+ T cell responses has thus far not been reported.
To characterize EBV-specific CD4+ T cell responses from acute infection through latency, short in vitro stimulation assays followed by intracellular cytokine staining were performed to measure the frequency of virus-specific CD4+ T cells. This method allows enumeration of Ag-specific T cells without relying on long-term in vitro culture techniques. Using this assay, CD4+ T cell responses to lytic and latent proteins were evaluated to determine the timing, magnitude, and specificity of EBV-specific CD4+ T cells over the course of infection. Peptide-based assays were then used for concurrent analysis of the timing, magnitude, and specificity of BZLF-1-specific CD4+ and CD8+ T cell responses. The relationship between EBV-specific CD4+ T cell responses and viral load during AIM was also evaluated.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Individuals presenting to the clinic at the University of Massachusetts at Amherst Student Health Service (Amherst, MA) with clinical symptoms consistent with AIM were recruited for this study. Following informed consent, 50 ml of blood was collected at each of five approximate time points: at presentation with symptoms (V1) and 3 wk (V2), 6 wk (V3), 6 mo (V4), and 1 year (V5) thereafter. Acute EBV infection was confirmed by the detection of IgM Abs to the EBV viral capsid Ag in patient sera.
Healthy EBV-seropositive and -seronegative donors were also recruited for the study. EBV-seropositive individuals experienced primary EBV infection at least 10 years before this study. EBV serological status was determined by the presence or absence of IgG Abs to the viral capsid Ag in serum. Following informed consent, blood (
10 ml) was collected periodically from these donors. These studies were approved by the Human Studies Committee at the University of Massachusetts Medical School (Worcester, MA).
Synthesis of GST-fusion proteins
The plasmid encoding GST-tagged BZLF-1 was generously provided by S. Kenney (University of North Carolina, Chapel Hill, NC). Genes encoding BMLF-1 (aa 1-438), EBNA-1 (aa 363-641), and EBNA-3A (aa 403-812) were PCR-amplified from B95.8 using the following primer sets: 5'-CGCGGGATCCCCATGGAGGGCAGCGAAGAA-3' and 5'-CGCGCAATTGTTATTGATTTAATCCAGGAAC-3' for BMLF-1; 5'-CGCGGGATCCCCGGGGGAAGTCGTGAAAGA-3' and 5'-CGCGGAATTCTTACTCCTGCCCTTCCTCACCC-3' for EBNA-1; and 5'-CGCGGGATCCCCGTAGAACCCGTCCCTGTC-3' and 5'-CGCGGAATTCTTAGGCCTCATCTGGAGGATC-3' for EBNA-3A. The amplified genes were cut by appropriate restriction enzymes and ligated into the multiple cloning site of the GST expression vector pGEX-3X (Amersham Pharmacia Biotech, Piscataway, NJ). Plasmids were used to transform Escherichia coli strain BL21DE3RP (Stratagene, La Jolla, CA). This strain was also transformed with the pGEX-3X plasmid containing no insert for purification of GST. Transformed clones were grown to midlog phase at 37°C and induced with 0.6 mM isopropyl-
-thiogalactosidase for 2–3 h. Bacteria were resuspended in buffer containing protease inhibitor (50 mM Tris-HCl, 25% sucrose, 1 mM EDTA, 0.2 mM PMSF, pH 8) and lysed by the sequential addition of 1 mg/ml lysozyme, 2 mg/ml DNase I, 1 M MgCl2, and lysis buffer (50 mM Tris-HCl (pH 8), 1% Triton X-100, 100 mM NaCl, 1% sodium deoxycholate). Lysates were snap-frozen in liquid N2, clarified after thawing by centrifugation at 17,400 x g, and incubated with glutathione Sepharose 4B (Amersham Pharmacia Biotech) overnight at 4°C.
GST and GST-fusion proteins were purified according to manufacturer’s instructions using a batchwise method with small modifications. Suspensions were centrifuged to sediment the matrix, and the supernatants were removed. The glutathione Sepharose 4B was washed with cold PBS until OD280 of the wash buffer was <0.010. Protein was eluted with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8), and PMSF was added at a final concentration of 0.1 mM. Protein from pooled eluates was quantified using Bradford assay and analyzed by SDS-PAGE and Western blot. Protein preparations were further purified over polymixin B columns (Pierce, Rockford, IL) to remove endotoxin according to manufacturer’s instructions. Concentrated stocks were stored at -20°C. Working stocks for immunology assays were diluted to 1 mg/ml in RPMI 1640 and stored at -20°C.
Intracellular cytokine staining for the detection and enumeration of EBV-specific CD4+ T cells
Ag stimulation of whole blood followed by intracellular staining for cytokine production was performed as previously described (11) with minor modifications. Briefly, heparinized, fresh whole blood or cryopreserved PBMC (0.5 ml) was incubated with 20 µg/ml GST or GST-fusion protein and 3 µg/ml each anti-CD28 and anti-CD49d (BD PharMingen, San Diego, CA) at 37°C for a total of 6 h. As a positive control, staphylococcal enterotoxin B (SEB) (Toxin Technology, Sarasota, FL) at 2 µg/ml was used. GolgiPlug (BD PharMingen) was added for the final 4 h of incubation. After incubation, EDTA was added at 2 mM, and the cells were vortexed repeatedly for 15 min. Red cells were lysed using FACS lysing solution (BD Biosciences, San Diego, CA) and washed in PBS containing 1% FCS. Cells were then made permeable in FACSPerm (BD Biosciences), washed, and stained with a mixture of anti-CD4-PerCP, anti-CD69-PE (both BD Biosciences), anti-CD45RO-FITC, and anti-IFN-
-APC (both BD PharMingen).
Cytokine-secreting cells were analyzed by four-color flow cytometry using FACSort with an added laser and CellQuest software (BD Biosciences). Isotype control Abs and appropriate positive and negative controls were used to define cytokine-secreting populations. Approximately 50,000–100,000 events were collected through the lymphocyte gate as determined by forward vs side scatter pattern. For cytokine secretion analysis, IFN- was plotted vs CD69 using a CD4+CD45RO+ gate. No cytokine production was detected by CD4+CD45RO- cells in our assays. Frequencies of IFN--secreting cells in response to GST-fusion proteins that were 2 SD above the mean background response to GST alone were considered significant (>0.06%). Cytokine production was not detected in response to EBV proteins in EBV-seronegative individuals. Comparable responder-cell frequencies were obtained using either fresh whole blood or cryopreserved cells as starting material.
Overlapping BZLF-1 peptides and intracellular cytokine staining
Sixteen 25-mer, overlapping (10 aa) peptides spanning the BZLF-1 protein were synthesized (Peptide Core Facility, University of Massachusetts Medical School). Peptides were dissolved in DMSO, and working stocks were further diluted in RPMI 1640 to a final concentration of 1 µg/µl. Peptide stimulation followed by intracellular cytokine staining was performed as above using 10 µg/ml peptide. Because all patients were HIV-negative, the A2-restricted Gag epitope (SLYNTVATL) was used as a negative control. Cells were stained with a mixture of Abs: anti-CD4-PerCP, anti-CD8-FITC (Sigma-Aldrich, St. Louis, MO), anti-CD69-PE, and anti-IFN--APC. They were then analyzed by flow cytometry as above. However, frequencies of IFN--secreting cells were determined by plotting IFN- vs CD69 after gating on CD4+ or CD8+ populations. Frequencies of cytokine-secreting cells in response to BZLF-1 peptides that were 2 SD above the mean background response to the A2-Gag peptide were considered significant (>0.03% for CD4+ T cells, >0.04% for CD8+ T cells).
Real-time PCR quantification of EBV viral load
EBV DNA copy frequency was estimated using a modification of the previously described real-time TaqMan PCR method for EBV quantification (12). B lymphocytes were enriched either from whole blood using the RosetteSep protocol that features tetrameric Ab complex technology (StemCell Technologies, Vancouver, British Columbia, Canada) or from freshly thawed PBMC by MACS using a B cell isolation kit (Miltenyi Biotec, Auburn, CA). Genomic DNA was extracted using the DNeasy kit (Qiagen, Valencia, CA). The oligonucleotide primer-pairs used in the reaction amplify conserved sequences of the BALF5 region that encode the viral DNA polymerase within the EBV genome, and their sequences are as follows: forward primer 5'-CGGAAGCCCTCTGGACTTC-3'; reverse primer 5'-CCCTGTTTATCCGATGGAATG-3'. A fluorogenic probe (5'-TGTACACGCACGAGAAATGCGCC-3'), which hybridizes to a sequence located within the region amplified by the forward and reverse primers, was synthesized with a FAM reporter molecule attached to the 5' end and a TAMRA quencher linked at the 3' end. The PCR mixture contained either 100 or 200 ng of genomic DNA in a 50-µl reaction volume containing 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 5 mM MgCl2; 10 mM EDTA; 100 µM each of dATP, dCTP, dGTP, and dTTP; 0.2 mM of each primer; 0.1 mM fluorogenic probe; and 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). The amplification was performed in a TaqMan 7700 Sequence Detector thermocycler (Applied Biosystems) according to the following cycling parameters: 10 min at 95°C to activate the AmpliTaq Gold DNA polymerase, then 45 cycles of 15 s at 95°C and 1 min at 62°C. DNA extracted from B cells from EBV-seropositive and -seronegative donors were included as positive and negative controls, respectively.
Serially diluted control plasmid (from 105 to 10 copies) containing a single copy of the BALF5 amplicon generated by the forward and reverse primers was included in each PCR assay. The threshold cycle (CT) value, corresponding to the point at which the real-time fluorescence exceeded 10x SD of the baseline for each sample, was calculated for each sample. The CT values were then plotted against the copy number to construct the standard curve, and from the CT values of test samples the EBV copy number was calculated using the software for data analysis (Sequence Detector version 1.6; Applied Biosystems). The results were expressed as copies of EBV genome per microgram of DNA. We have modified the assay to determine the cellular equivalents of template DNA within each test sample. To this end, real-time PCR was performed on aliquots of template DNA to amplify a region within the duplicate-copy cellular chemokine receptor gene, CCR5. The primers used were 5'-GCTGTGTTTGCGTCTCTCCCAGGA-3' (forward) and 5'-CTCACAGCCCTGTGCCTCTTCTTC-3' (reverse), and the corresponding fluorogenic oligonucleotide probe used was 5'FAM-AGCAGCGGCAGGACCAGCCCCAAG-TAMRA-3'. Serially diluted plasmid containing a single copy of the CCR5 gene was used to generate the standard curve, and the CCR5 copy number in the sample was determined. The number of cellular equivalents was then calculated, and the EBV copy frequency in the population of B lymphocytes in each test sample was expressed as copies of EBV genome per 106 B cells. Each sample was tested in triplicate, and the mean of the three values was considered for quantifying the EBV load. Samples with CT values that exceeded 45 cycles in our conditions of the assay were considered negative for EBV.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We used intracellular cytokine assays to detect and measure the frequency of EBV-specific CD4+ T cells in the peripheral blood of individuals at presentation with AIM and afterward. Because we (4) and others (3, 5) have previously shown that EBV lytic (BZLF-1, BMLF-1) and latent (EBNA-3A) proteins are commonly recognized by CD8+ T cells, and because recognition of EBNA-1 by CD4+ T cells has recently been reported (7, 10), we prioritized these proteins for study. The frequencies of EBV-specific CD4+ T cells were measured using intracellular IFN- staining following in vitro stimulation with each soluble protein (Fig. 1a); SEB was used as the positive control in these studies. Cytokine production was not detected following the in vitro stimulation of whole blood from healthy, EBV-seronegative adults (Fig. 1b).
|
|
|
In summary, EBV lytic and latent protein-specific CD4+ T cell responses were commonly detected at presentation with AIM. Responses to BZLF-1 were most commonly detected and were generally of higher frequencies than responses to the other proteins. Most individuals responded to two or more proteins.
EBV-specific CD4+ T cell responses decline rapidly following acute infection
Blood was collected at sequential time points up to 1 year following presentation with AIM to determine the stability of EBV-specific CD4+ T cell responses over time following acute EBV infection. As shown in Table I and Fig. 2, the number of individuals responding and the responder-cell frequencies of BZLF-1-, BMLF-1-, EBNA-1-, and EBNA-3A-specific CD4+ T cells were highest within 3 wk of presentation with AIM and declined rapidly over time following primary infection. By 6 wk following presentation, BZLF-1 responses were detected in only 61% of individuals, BMLF-1 and EBNA-3A responses in only 33% of individuals, and EBNA-1 responses in only 22% of individuals (Table I). Median responder-cell frequencies in individuals with detectable responses dropped to 0.12, 0.07, 0.09, and 0.09% for BZLF-1, BMLF-1, EBNA-1, and EBNA-3A, respectively, by the 6-wk time point. EBV-specific CD4+ T cells were undetectable in most individuals by 6 mo and in all individuals by 1 year following AIM. It was of interest that EBV-specific responses were not detected in any of 12 EBV-seropositive healthy adults tested (data not shown).
To determine whether other cytokines were produced in response to these EBV Ags over the course of infection, we assayed for IL-4 production by intracellular cytokine staining and by ELISPOT assay. IL-4 production was not detected by intracellular cytokine staining or ELISPOT assay in response to in vitro stimulation with BZLF-1, BMLF-1, EBNA-1, or EBNA-3A proteins at any time point from presentation through convalescence (data not shown).
Differences in the timing, frequency, and specificity of BZLF-1-specific CD4+ and CD8+ T cell responses over time following infection
In the murine lymphocytic choriomeningitis virus (LCMV) model, CD4+ and CD8+ T cell effectors appear to be important for clearance of chronic viral infection (13), and CD4+ T cell help appears necessary for the maintenance of CD8+ T cell memory (14). Therefore, we were interested in examining potential relationships between the timing, frequency, and specificity of BZLF-1-specific CD4+ and CD8+ T cell responses. Intracellular cytokine staining was performed after stimulation with overlapping peptides spanning the BZLF-1 protein, to determine the region of the protein recognized by each T cell subset within an individual over the course of AIM. Peptide sequences and individual CD4+ or CD8+ T cell responses to these peptides are shown in Table II.
|
|
|
In summary, BZLF-1-specific CD4+ T cell responses tended to peak earlier and at lower frequencies than BZLF-1-specific CD8+ T cell responses and were not persistently detectable in most individuals 6 wk or more following presentation with AIM. Within an individual, CD4+ and CD8+ T cell responses targeted different regions of the BZLF-1 protein.
EBV-specific CD4+ T cell responses correlate with blood EBV viral load over the course of infection
To examine the relationship between EBV load and EBV-specific CD4+ T cell responses, viral load was quantitated over the course of AIM using real-time PCR (Fig. 4). We found that EBV load was highest at presentation with AIM (range = 1.13 x 104 to 2.28 x 106 copies/106 B cells; median = 1.47 x 105 copies/106 B cells; n = 9). To determine whether EBV load at presentation correlated with Ag-specific CD4+ T cell frequency, log viral load was plotted against the frequency of CD4+ T cells specific for BZLF-1, BMLF-1, EBNA-1, and EBNA-3A (Fig. 5). While BMLF-1-, EBNA-1-, and EBNA-3A-specific CD4+ T cell frequency correlated with EBV load at presentation (R = 0.56, 0.45, and 0.59, respectively), only a weak correlation was observed between viral load and BZLF-1-specific CD4+ T cell frequency (R = 0.34). However, it should be noted that one patient (E-1122) appeared to be an outlier; upon exclusion of this patient, reanalysis of the data revealed a good correlation between viral load and EBV-specific CD4+ T cell frequency at presentation with AIM (R = 0.63, 0.55, 0.54, and 0.59 for BZLF-1, BMLF-1, EBNA-1, and EBNA-3A, respectively).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The marked lymphocytosis observed during AIM involves an expansion of both CD4+ and CD8+ T cells (16). It has recently been shown that the expansion of CD8+ T cells is largely Ag-specific (3, 4). The recent discovery of an EBV-induced superantigen suggests that the CD4+ T cell expansion may be, in part, nonspecifically driven (17). However, our data document that up to 2.7% of circulating effector/memory CD4+ T cells are EBV-specific. Moreover, this response is broadly specific in most individuals and is directed at both lytic and latent EBV Ags. To our knowledge, this is the first ex vivo demonstration of the Ag-specificity of CD4+ T cells during primary EBV infection.
CD4+ T cell recognition of both lytic and latent proteins during acute EBV infection contrasts with the preferential recognition of lytic proteins by CD8+ T cells (3, 4, 5). Latent protein-specific CD8+ T cells are infrequently detected at presentation with AIM but become detectable after a few weeks. Some have suggested that the differences in the pattern of responses to EBV proteins over time are due to differences in the timing of expression of these proteins. Our data demonstrating the detection of CD4+ T cells specific for both lytic and latent proteins at presentation with AIM suggest that the predominance of lytic-specific over latent-specific CD8+ T cells during acute EBV infection cannot be accounted for simply by differences in Ag expression. It is more likely that they reflect differences in the availability of these proteins to the appropriate APCs or processing pathways for CD4+ and CD8+ T cells during acute infection.
By examining both CD4+ and CD8+ T cell responses against BZLF-1 during AIM, we demonstrated differences in the expansion and stability of BZLF-1-specific CD4+ and CD8+ T cells. Measured frequencies of BZLF-1-specific CD4+ T cells were highest at presentation, but the rate of detection and the frequencies of detectable BZLF-1-specific CD4+ T cells declined over the first 6 wk of study. EBV-specific CD4+ T cell frequencies generally paralleled peripheral blood viral load, a finding that is consistent with the greater dependence of CD4+ T cell proliferation on Ag exposure in vitro (18, 19). By contrast, BZLF-1-specific CD8+ T cell frequencies continued to increase in most individuals over the weeks following presentation and remained detectable during convalescence. Whereas the frequencies of BZLF-1-specific CD8+ T cells correlated with blood viral load at presentation with AIM (data not shown), they subsequently increased in most individuals as viral load declined, suggesting that the expansion and maintenance of the CD8+ T cell population do not require persistently high levels of Ag (20).
Our findings bear some similarities to but also important differences from the results of studies of acute viral infections in mice. Murine studies of LCMV infection have demonstrated three general phases of T cell responses (reviewed in Ref. 21). During the expansion phase, the clonal expansion of virus-specific CD8+ T cells appears to precede and exceed that of virus-specific CD4+ T cells. Following clearance of LCMV (a nonpersistent virus), virus-specific CD8+ T cells undergo a more pronounced reduction in frequency than virus-specific CD4+ T cells (contraction phase). After the contraction phase, virus-specific CD8+ T cell frequencies appear to be stable, while virus-specific CD4+ T cell frequencies decline (22). Our data are similar to the murine LCMV data in that they demonstrate a less-robust expansion of virus-specific CD4+ T cells than CD8+ T cells, which is likely related to the slower rate of CD4+ T cell expansion documented in in vitro and in vivo studies (21). However, it is of interest that EBV-specific CD4+ T cell responses appear to be less stable than those generated during nonpersistent infection with LCMV.
The early detection of EBV-specific CD4+ T cells during acute EBV infection in this study is consistent with a previous study documenting the early detection of CMV-specific CD4+ T cells during primary CMV infection in kidney transplant recipients (23). Frequencies of CMV-specific CD4+ T cells peaked very early (median = 7 day) after detection of CMV DNA and declined rapidly thereafter. However, CMV-specific CD4+ T cells were readily detected in the peripheral blood of convalescent individuals (11, 23) even though CMV DNA was not commonly detected in the peripheral blood of healthy CMV carriers (24). Differences in the biology of these persistent viruses, such as the location and degree of Ag expression or the availability of Ag for processing and presentation, may strongly influence the virus-specific memory T cell pool.
There are many potential reasons that we were unable to detect EBV-specific CD4+ T cells following short-term in vitro stimulation during viral latency. First, these cells may be sequestered at sites of viral reactivation and replication such as the tonsil. Experiments using cells from tonsillar tissue are currently under way to address this possibility. Second, the frequency of memory EBV-specific CD4+ T cells may be below the limits of detection of even the newer, more sensitive assays used in this study. This possibility is supported by several published studies reporting the detection of EBV-specific CD4+ T cells during EBV latency (6, 7, 8, 10, 25), many of which required the generation of EBV-specific CD4+ T cell lines from healthy EBV carriers by long-term in vitro culture. Recently, Paludan et al. (26) reported detection of EBNA-1-specific CD4+ T cells in the peripheral blood of a small number of healthy EBV carriers by intracellular cytokine staining at frequencies ranging from 0.24 to 0.66% of CD4+ T cells. However, we have been unable to replicate these findings despite the fact that our EBNA-1 construct (aa 363-641), like their EBNA-1 construct (aa 458-641), contains defined CD4+ T cell epitopes.
In conclusion, EBV lytic- and latent-specific CD4+ T cells are commonly detected during early EBV infection but differ in their kinetics and specificity from EBV-specific CD8+ T cell responses. A better understanding of how these Ag-specific cells contribute to the resolution of acute infection will not only elucidate the role of virus-specific CD4+ T cells in persistent human viral infections, but may also aid in developing vaccination strategies as well as potential treatments for EBV-associated malignancies.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Katherine Luzuriaga, Pediatrics/Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Biotech II, Suite 318, Worcester, MA 01605. E-mail address: katherine.luzuriaga{at}umassmed.edu
3 Abbreviations used in this paper: AIM, acute infectious mononucleosis; EBNA, Epstein-Barr nuclear Ag; SEB, staphylococcal enterotoxin B; CT, threshold cycle; LCMV, lymphocytic choriomeningitis virus.
Received for publication October 4, 2002. Accepted for publication December 19, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Liu, H.-E. Claesson, Y. Mahshid, G. Klein, and E. Klein Leukotriene B4 activates T cells that inhibit B-cell proliferation in EBV-infected cord blood-derived mononuclear cell cultures Blood, March 1, 2008; 111(5): 2693 - 2703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bhaduri-McIntosh, M. J. Rotenberg, B. Gardner, M. Robert, and G. Miller Repertoire and frequency of immune cells reactive to Epstein-Barr virus-derived autologous lymphoblastoid cell lines Blood, February 1, 2008; 111(3): 1334 - 1343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Zaunders, W. B. Dyer, M. L. Munier, S. Ip, J. Liu, E. Amyes, W. Rawlinson, R. De Rose, S. J. Kent, J. S. Sullivan, et al. CD127+CCR5+CD38+++ CD4+ Th1 Effector Cells Are an Early Component of the Primary Immune Response to Vaccinia Virus and Precede Development of Interleukin-2+ Memory CD4+ T Cells. J. Virol., October 1, 2006; 80(20): 10151 - 10161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Zaunders, S. Ip, M. L. Munier, D. E. Kaufmann, K. Suzuki, C. Brereton, S. C. Sasson, N. Seddiki, K. Koelsch, A. Landay, et al. Infection of CD127+ (Interleukin-7 Receptor+) CD4+ Cells and Overexpression of CTLA-4 Are Linked to Loss of Antigen-Specific CD4 T Cells during Primary Human Immunodeficiency Virus Type 1 Infection. J. Virol., October 1, 2006; 80(20): 10162 - 10172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Parra-Lopez, J. M. Calvo-Calle, T. O. Cameron, L. E. Vargas, L. M. Salazar, M. E. Patarroyo, E. Nardin, and L. J. Stern Major Histocompatibility Complex and T Cell Interactions of a Universal T Cell Epitope from Plasmodium falciparum Circumsporozoite Protein J. Biol. Chem., May 26, 2006; 281(21): 14907 - 14917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Adhikary, U. Behrends, A. Moosmann, K. Witter, G. W. Bornkamm, and J. Mautner Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins J. Exp. Med., April 17, 2006; 203(4): 995 - 1006. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Williams and D. H. Crawford Epstein-Barr virus: the impact of scientific advances on clinical practice Blood, February 1, 2006; 107(3): 862 - 869. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Landais, X. Saulquin, M. Bonneville, and E. Houssaint Long-Term MHC Class II Presentation of the EBV Lytic Protein BHRF1 by EBV Latently Infected B Cells following Capture of BHRF1 Antigen J. Immunol., December 15, 2005; 175(12): 7939 - 7946. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Piriou, K. van Dort, N. M. Nanlohy, M. H. J. van Oers, F. Miedema, and D. van Baarle Loss of EBNA1-specific memory CD4+ and CD8+ T cells in HIV-infected patients progressing to AIDS-related non-Hodgkin lymphoma Blood, November 1, 2005; 106(9): 3166 - 3174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Stone, W. E. Demkowicz Jr., and L. J. Stern HLA-restricted epitope identification and detection of functional T cell responses by using MHC-peptide and costimulatory microarrays PNAS, March 8, 2005; 102(10): 3744 - 3749. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Piriou, K. van Dort, N. M. Nanlohy, F. Miedema, M. H. van Oers, and D. van Baarle Altered EBV Viral Load Setpoint after HIV Seroconversion Is in Accordance with Lack of Predictive Value of EBV Load for the Occurrence of AIDS-Related Non-Hodgkin Lymphoma J. Immunol., June 1, 2004; 172(11): 6931 - 6937. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Zaunders, W. B. Dyer, B. Wang, M. L. Munier, M. Miranda-Saksena, R. Newton, J. Moore, C. R. Mackay, D. A. Cooper, N. K. Saksena, et al. Identification of circulating antigen-specific CD4+ T lymphocytes with a CCR5+, cytotoxic phenotype in an HIV-1 long-term nonprogressor and in CMV infection Blood, March 15, 2004; 103(6): 2238 - 2247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Gibson, G. Piccinini, D. Lilleri, M. G. Revello, Z. Wang, S. Markel, D. J. Diamond, and K. Luzuriaga Human Cytomegalovirus Proteins pp65 and Immediate Early Protein 1 Are Common Targets for CD8+ T Cell Responses in Children with Congenital or Postnatal Human Cytomegalovirus Infection J. Immunol., February 15, 2004; 172(4): 2256 - 2264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Landais, X. Saulquin, E. Scotet, L. Trautmann, M.-A. Peyrat, J. L. Yates, W. W. Kwok, M. Bonneville, and E. Houssaint Direct killing of Epstein-Barr virus (EBV)-infected B cells by CD4 T cells directed against the EBV lytic protein BHRF1 Blood, February 15, 2004; 103(4): 1408 - 1416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Amyes, C. Hatton, D. Montamat-Sicotte, N. Gudgeon, A. B. Rickinson, A. J. McMichael, and M. F.C. Callan Characterization of the CD4+ T Cell Response to Epstein-Barr Virus during Primary and Persistent Infection J. Exp. Med., September 15, 2003; 198(6): 903 - 911. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
