Non-mass versus mass-like ultrasound patterns in ductal carcinoma in situ: is there an association with high-risk histology?

AIM: To review the ultrasound (US) patterns of pure ductal carcinoma in situ (DCIS) using a non-mass-like (NML) versus mass-like (ML) classification and to investigate histopathological associations. MATERIALS AND METHODS: The present study was a retrospective analysis of sonographically evident pure DCIS lesions detected in a mammographic (MG) screening programme over a 7-year period from 2008. All lesions had undergone US-guided 14 G core biopsies with no upgrades to invasive disease on surgical histopathology. Lesions that were three-dimensional with convex margins were classified as ML and all others as NML. ML lesions were subdivided into solid, cystic, or mixed, and NML lesions into ductal and non-ductal. Imaging and pathological characteristics of NML versus ML lesions were investigated using logistic regression. RESULTS: There were 78 lesions in 75 participants. NML lesions accounted for 45 (58%) lesions, comprising 27 (60%) ductal and 18 (40%) non-ductal subtypes. There were 33 (42%) ML lesions; the largest subgroup being solid (n=21, 64%). Significant associations between lesion type and lesion size on US (<15 versus ≥15 mm), presence of US and mammographic calcification and posterior shadowing on sonography were identified. NML lesions had fivefold higher odds (OR=5.41 95% confidence interval [CI]: 2.03, 14.39, p=0.001) to be high grade and sevenfold higher odds (OR=7 95% CI: 1.75, 27.99, p=0.006) to have comedo necrosis on histopathology. CONCLUSION: DCIS lesions can be successfully classified using ML and NML lesion descriptors and NML morphology on US is associated with histological features of "high-risk" DCIS.

Neutrophil activation during acute human anaphylaxis: analysis of MPO and sCD62L.

BACKGROUND: The mechanisms involved in the amplification of the mast cell response during anaphylaxis are unclear. Mouse models of anaphylaxis demonstrate the critical involvement of neutrophils. These innate immune cells are highly abundant in peripheral blood and can be rapidly activated to trigger both local and systemic inflammation. OBJECTIVE: To investigate neutrophil activation in peripheral blood during acute human anaphylaxis. METHODS: Patients presenting to the emergency department with anaphylaxis underwent blood sampling upon enrolment and at up to three subsequent time-points. Traditional anaphylaxis biomarkers, histamine and mast cell tryptase, were measured by ELISA and ImmunoCAP, respectively. Plasma myeloperoxidase concentrations were measured by ELISA, serum soluble CD62L concentrations by cytometric bead array, and both compared to healthy controls. RESULTS: In 72 patients, 37 (51%) had severe anaphylaxis, 33 (60%) were histamine positive, and 47 (70%) were mast cell tryptase positive. At enrolment, myeloperoxidase concentrations were 2.9- (95% CI: 1.3, 6.5) and 5.0- (95% CI: 2.4, 10.5) fold higher in moderate and severe patients, respectively, compared with healthy controls, and remained stable over the first 5 h following symptom onset. At enrolment, soluble CD62L was 29% (95% CI: 19, 38) and 31% (95% CI: 22, 40) lower in moderate and severe patients, respectively, than healthy controls, and was stable over the first 5 h. There were no associations between myeloperoxidase or soluble CD62L concentrations and either histamine or mast cell tryptase concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: These results provide compelling evidence for the involvement of neutrophils during acute human anaphylaxis, suggesting they are activated early in the reaction, regardless of mast cell activation. This important finding increases our understanding of the basic mechanisms of anaphylaxis, a necessary precursor to improving treatment and prevention.

Effect of expiratory positive airway pressure on tidal volume during non-invasive ventilation.

During non-invasive ventilation (NIV), tidal volume ( Vt) will depend upon the difference between inspiratory and expiratory positive airway pressure (IPAP and EPAP, respectively), provided the respiratory muscles are relaxed and the lungs and chest wall therefore move along their passive pressure-volume curves. To test this hypothesis, we studied the effect of increasing EPAP during pressure-controlled modes of NIV in 30 long-term ventilator users (10 each with scoliosis, obesity hypoventilation or neuromuscular disorders). While maintaining the same IPAP, addition of 5 cmH2O of EPAP reduced mean Vt by 167 ml; 10 cmH2O reduced Vt by 367 ml. This pattern was seen in all three patient groups. EPAP has several potential advantages, for example maintaining upper airway patency, preventing basal atelectasis and facilitating triggering. EPAP does, however, appear to reduce Vt. Decreasing EPAP is an alternative to increasing IPAP if measurements of gas exchange during NIV indicate that ventilation is inadequate.

Long-term non-invasive ventilation in muscular dystrophy.

Long-term non-invasive ventilation (NIV) was introduced in the 1980s, initially mainly for patients with poliomyelitis, muscular dystrophy (MD) or scoliosis. The obesity-hypoventilation syndrome has since become the commonest reason for referral to most centres providing home-NIV. Patients with MD are numerically a much smaller part of the workload, but as their disease progresses the need for ventilatory support changes and they require regular comprehensive assessment of their condition. We have examined the trend in MD use of home-NIV in our unit over the last 25 years. The number of new referrals appears to be stabilizing at around 20-25 over a 5-year period, equivalent to approximately 0.5 per 100,000 of population per year. The mean age at commencement of home-NIV is now 37.5 years, with 5-year survival rates of 70-75%. Ten-year survival rates are just over 40%. The distance of usual place of residence from our unit is fairly stable, currently at a mean of 27 km. Excellent survival rates mean that patients with MD, while numerically small, are likely to remain an important part of the workload of centres providing home-NIV. Our data should prove useful in the planning of future services for this group of patients.

The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

Retrospective preoperative assessment of the axillary lymph nodes in patients with breast cancer and literature review.

AIM: To evaluate the accuracy of axillary ultrasound, compared with published literature, identify women at low risk for lymph node (LN) involvement, and to determine which clinical, pathological, and imaging findings best predict LN involvement. MATERIAL AND METHODS: From June 2010 to April 2012, 288 women with breast lesions that were suspicious of malignancy (category 4) or malignant (category 5) underwent axillary ultrasound examination. A 3 mm LN cortical thickness was used as the threshold to prompt fine-needle aspiration biopsy (FNAB) of the LN. Data were gathered regarding size, site, and grade of the index breast lesion and cortical thickness of the LN. RESULTS: Using a cut-off point of <3 mm versus ≥3 mm, abnormal cortical thickness had a sensitivity and specificity of 56.3% and 86.7%, respectively. Breast cancer size was significantly associated with the odds of LN metastasis (p<0.001). There were 69 patients with breast cancers of ≤10 mm and 18% had positive axillary LNs. A much higher rate of malignancy was observed in breast cancers located in multiple sites and in a central location. CONCLUSION: The likelihood of axillary LN metastasis increases with cortical thickness ≥3 mm and this concurs with the literature. A low-risk group of women was identified with screen-detected, low-grade small cancers with LNs with a cortical thickness of <3 mm. Additional features other than cortical thickness >3 mm (such as shape [rounding], echogenicity [markedly hypo-echoic cortex], and morphology [hilar compressional displacement, loss of echogenic outer capsule and angular margins]) should be used to indicate FNAB in patients with a palpable lump, multiple or central cancers, and cancers >20 mm.

High frequency of herpesvirus-specific clonotypes in the human T cell repertoire can remain stable over decades with minimal turnover.

High-throughput T cell receptor sequencing on sequentially banked blood samples from healthy individuals has shown that high-frequency clonotypes can remain relatively stable for up to 18 years, with minimal inflation, deflation, or turnover. These populations included T cell expansions specific for Epstein-Barr virus. Thus, in spite of exposure to a barrage of microorganisms over the course of life, the dominant clonotypes in the mature peripheral T cell repertoire can alter surprisingly little.

EBV & HHV6 reactivation is infrequent and not associated with MS clinical course.

BACKGROUND: Among the environmental factors associated with multiple sclerosis (MS) causation, some of the strongest associations are with Epstein-Barr virus (EBV), and to a lesser extent human herpesvirus 6 (HHV6). Associations with clinical course are less conclusive, however. METHODS: We evaluated serum anti-EBV-EA-R IgG and anti-HHV6 IgM, and EBV and HHV6 viral load (VL) for their associations with relapse, disability, and progression in disability in a prospective cohort of 198 participants with clinically definite MS. RESULTS: Anti-EBV-EA-R IgG was detected in 81.8% of cases at study entry, and titers remained essentially unchanged during the study. Anti-HHV6 IgM was detected in only one participant, and EBV-VL (29%) and HHV6-VL (1.8%) were detected in a minority of samples, and where detected levels were low. Our previously demonstrated association between anti-HHV6 IgG and relapse hazard was not affected by adjustment for parameters of reactivation. We found no evidence that any of the viral markers were associated with disability or progression in disability. In relation to relapse, only EBV-VL was positively associated, although this was strongly influenced by a single individual. CONCLUSION: Using a prospective cohort design, we found no convincing evidence that reactivation parameters of EBV or HHV6 were associated with subsequent MS relapse hazard or progression in disability, confirming previous findings, and indicating that herpesvirus reactivation is not an important driver of relapse or disability in this established MS population.

Laser tongue base mucosectomy is a useful diagnostic tool in the management of unknown primary cancers of the head and neck region.

OBJECTIVE: Tongue base mucosectomy identified cancer in 78 per cent of cancers of unknown primary in a recent meta-analysis. The carbon dioxide laser is an alternative technique if there is no access to a robot. This study aimed to describe the steps for undertaking tongue base mucosectomy using the carbon dioxide laser and its diagnostic utility in cancers of unknown primary. METHOD: This was a prospective feasibility study utilising carbon dioxide laser for tongue base mucosectomy in cancers of unknown primary. Data collected included demographic data and p16 status. RESULTS: There were 14 cancers of unknown primary with 86 per cent p16 positivity on immunohistochemistry. Laser tongue base mucosectomy alone identified the cancer primary in 7 of 12 (58 per cent) cancers of unknown primary among p16 positive tumours and 0 of 2 (0 per cent) among p16 negative tumours. Combining bilateral tonsillectomy with laser tongue base mucosectomy resulted in identification of the primary cancer in 8 of 12 (67 per cent) p16 positive tumours. CONCLUSION: In centres without a robot, tongue base mucosectomy using the carbon dioxide laser is a viable alternative, especially in combination with bilateral tonsillectomy in p16 positive cases.

Does academic output correlate with better mortality rates in NHS trusts in England?

OBJECTIVE: To establish whether there is an association between academic output and mortality rates for National Health Service (NHS) trusts. METHODS: Hospital standardized mortality ratios were obtained from Dr Foster hospital report cards. The Medline database of biomedical citations was queried to establish the number of citations credited to each NHS trust and constituent hospitals from 2006 to 2010. Admissions totals for NHS trusts for 2009-2010 were obtained from Hospital Episode Statistics Online. The number of citations per admission was calculated and used as an indicator of academic output as this reflects the workload of the trust. RESULTS: Spearman's rank analysis was performed to identify any correlation between citations per admission and the inverse of four types of mortality rate: high-risk conditions, r = 0.20 (P = 0.01); low-risk conditions, r = -0.06 (P = 0.46); deaths after surgery, r = 0.193 (P = 0.019); and overall mortality, r = 0.291 (P < 0.01). CONCLUSION: The results of this preliminary study demonstrate a significant correlation between academic output and mortality rates. The correlation coefficients are small, but the findings of this study encourage further debate.

The effect of radiological imaging on treatment delay and hospitalisation in patients with peritoneal dialysis-related peritonitis: A secondary analysis of the PROMPT study.

BACKGROUND: In peritoneal dialysis-related peritonitis (peritonitis), delayed antibiotic therapy is associated with adverse outcomes. Identifying barriers to timely treatment may improve outcomes. AIM: To determine the impact of radiological investigations on treatment delay and predictors of hospitalisation and length of stay (LOS). METHODS: Retrospective review of patients with presumed peritonitis in Western Australia. RESULTS: In 153 episodes of peritonitis, 79 (51.6%) resulted in admission with a median LOS of 3 days (Q1, Q3: 1, 6). In a multivariable model, significant predictors of admission were abnormal exit-site (odds ration (OR) 5.7; 95% confidence interval (CI): 1.4, 23.6; p = 0.02), failure to detect a cloudy bag (OR 11.9; 95%CI: 3.2, 44.7; p < 0.001), female sex (OR 3.3; 95% CI: 1.4, 9.7; p = 0.027), radiological imaging within 24 h (OR 8.8; 95% CI: 2.2, 34.8; p = 0.002) and contact with ambulant care facility (OR 0.32, 95% CI: 0.11, 0.98; p = 0.04). Imaging within 24 h of presentation occurred in 41 (27%) episodes of peritonitis, mostly plain X-rays (91%), of which 83% were clinically irrelevant. Imaging performed within 24 h of presentation increased the median time to antibiotic treatment (2.9 h (Q1, Q3: 1.6, 6.4) vs 2.0 h (Q1, Q3: 1, 3.8; p = 0.046)). Imaging performed prior to administering antibiotics significantly increased the median time to treatment (4.7 h (Q1, Q3: 2.9, 25) vs 1.5 h (Q1, Q3: 0.75, 2.5; p < 0.001)) in those where imaging followed antibiotic treatment. CONCLUSIONS: Half of all presentations with peritonitis result in hospital admission. Radiological imaging was associated with an increased risk of hospitalisation, potentially contributes to treatment delay, and was mostly clinically unnecessary. When required, imaging should follow antibiotic therapy.

The correlation between cauda equina nerve root volume and sensory block height after spinal anaesthesia with glucose-free bupivacaine.

We examined the association between cauda equina nerve root volume and sensory block height in 15 patients undergoing spinal anaesthesia with 0.5% glucose-free bupivacaine. Magnetic resonance imaging and a separate image segmentation program were used to calculate the volume of the nerve roots. Nerve root volume was also correlated with lumbosacral cerebrospinal fluid volume and with patients' physical characteristics. Nerve root volume correlated negatively with sensory block height (Spearman rho -0.61 (95% CI -0.85 to -0.14)) and body mass index (Spearman rho -0.66 (95% CI -0.87 to -0.24)) but positively with cerebrospinal fluid volume (Spearman rho 0.76 (95% CI 0.43-0.91)). Factors that are thought to influence cerebrospinal fluid volume, such as body mass index, might similarly affect the volume of the nerve roots. The size of the nerve roots may influence the spread of spinal anaesthesia.

An alloresponse in humans is dominated by cytotoxic T lymphocytes (CTL) cross-reactive with a single Epstein-Barr virus CTL epitope: implications for graft-versus-host disease.

The phenomenon of T cell allorecognition is difficult to accommodate within the framework of a T cell repertoire positively selected in the thymus, unless allorecognition results from the cross-reactions of self-major histocompatibility complex restricted T cells. Herein, we demonstrate the dual specificity of cytotoxic T lymphocyte (CTL) clones for the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, presented on HLA-B8, and the alloantigen HLA-B*4402. CTL which recognized peptide FLRGRAYGL in association with HLA-B8 could be reactivated in vitro from healthy individuals who had been exposed previously to EBV, using stimulator cells expressing the cross-reacting alloantigen HLA-B*4402. Limiting dilution analysis of the alloresponse to HLA-B*4402 in eight healthy individuals revealed that HLA-B8+, EBV-sero+ donors had higher CTL precursor frequencies for alloantigen HLA-B*4402 than EBV-sero- control donors. It is surprising that the majority (65-100%) of anti-HLA-B*4402 CTL, generated in limiting dilution mixed lymphocyte reactions between responder cells from HLA-B8+, EBV-sero+ individuals and HLA-B*4402+ stimulators, also recognized the EBV CTL epitope FLRGRAYGL/HLA-B8. In contrast to previous studies showing extensive diversity in the T cell repertoire against individual alloantigens, these data demonstrate that the response to an alloantigen can be dominated by CTL cross-reactive with a single viral epitope, thus illustrating a possible mechanism for the frequent clinical association between herpesvirus exposure and graft-versus-host disease after bone marrow transplants.

An Epstein-Barr virus-specific cytotoxic T cell epitope in EBV nuclear antigen 3 (EBNA 3).

Epstein-Barr virus (EBV)-specific CTL clones were isolated that recognized A-type EBV transformants but not B-type transformants. These A-type-specific CTL clones (HLA B8 restricted) were used to screen peptides derived from the EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 as potential CTL epitopes. Of the 76 peptides screened, one sequence from EBNA 3 (residues 329-353) was recognized by A-type-specific CTL clones after absorption onto target cells (either autologous B-type transformants or PHA blasts). This report is the first description of an EBV target epitope recognized by specific CTL clones.

T cell-T cell killing is induced by specific epitopes: evidence for an apoptotic mechanism.

Epstein-Barr virus-specific cytotoxic T lymphocyte clones were shown to be an effective target for their own lysis when incubated in the presence of their specific epitopes but not in the presence of irrelevant epitopes. The mode of cell killing appeared to be by apoptosis and was prevented by previously described inhibitors of the process. Degranulation, as measured by serine esterase activity, was involved in this form of T cell-T cell killing. This is the first report of T cell-T cell killing by apoptosis and is only observed in the presence of a specific epitope. This result may be of significance in the use of peptide-based vaccines.

T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen.

Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self-reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402. Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination of TCR-binding residues.

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta chains from HLA-B8-restricted, CD8+ cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved beta chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.

Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.