Adoptive Transfer of EBV Specific CD8+ T Cell Clones Can Transiently Control EBV Infection in Humanized Mice

Epstein Barr virus (EBV) infection expands CD8⁺ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8⁺ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8⁺ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8⁺ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development.     

Antiretroviral therapy reduces the magnitude and T cell receptor repertoire diversity of HIV-specific T cell responses without changing T cell clonotype dominance

After initiation of antiretroviral therapy (ART), HIV loads and frequencies of HIV epitope-specific immune responses decrease. A diverse virus-specific T cell receptor (TCR) repertoire allows the host to respond to viral epitope diversity, but the effect of antigen reduction as a result of ART on the TCR repertoire of epitope-specific CD8(+) T cell populations has not been well defined. We determined the TCR repertoires of 14 HIV-specific CD8(+) T cell responses from 8 HIV-positive individuals before and after initiation of ART. We used multiparameter flow cytometry to measure the distribution of memory T cell subsets and the surface expression of PD-1 on T cell populations and T cell clonotypes within epitope-specific responses from these individuals. Post-ART, we noted decreases in the frequency of circulating epitope-specific T cells (P = 0.02), decreases in the number of T-cell clonotypes found within epitope-specific T cell receptor repertoires (P = 0.024), and an overall reduction in the amino acid diversity within these responses (P < 0.0001). Despite this narrowing of the T cell response to HIV, the overall hierarchy of dominant T cell receptor clonotypes remained stable compared to that pre-ART. CD8(+) T cells underwent redistributions in memory phenotypes and a reduction in CD38 and PD-1 expression post-ART. Despite extensive remodeling at the structural and phenotypic levels, PD-1 was expressed at higher levels on dominant clonotypes within epitope-specific responses before and after initiation of ART. These data suggest that the antigen burden may maintain TCR diversity and that dominant clonotypes are sensitive to antigen even after dramatic reductions after initiation of ART.     

High-Throughput Sequencing of Islet-Infiltrating Memory CD4+ T Cells Reveals a Similar Pattern of TCR Vβ Usage in Prediabetic and Diabetic NOD Mice

Autoreactive memory CD4⁺ T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRβ repertoire of the memory CD4⁺ T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRβ repertoire of sorted islet-infiltrating memory CD4⁺CD44^high T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4⁺CD44^high T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4⁺CD44^high TCRβ repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vβ2), TRBV13-3 (Vβ8.1), and TRBV19 (Vβ6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRβ might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes.     

Changing patterns of dominant TCR usage with maturation of an EBV-specific cytotoxic T cell response

Infection with EBV provides a unique opportunity to follow the human CD8(+) T cell response to a persistent, genetically stable agent from the primary phase, as seen in infectious mononucleosis (IM) patients, into long-term memory. This study focuses on the response to an immunodominant HLA-A2.01-restricted epitope, GLCTLVAML, from the EBV-lytic cycle Ag BMLF1. TCR analysis of the highly amplified primary response to this epitope revealed markedly oligoclonal receptor usage among in vitro-derived clones, with similar clonotypes dominant in all three IM patients studied. Direct staining of IM T cell preparations with the A2.01/GLCTLVAML tetramer linked this oligoclonal epitope-specific response with appropriate Vbeta subset expansions in the patients' blood. These patients were studied again >2 years later, at which time TCR analysis of in vitro-reactivated clones suggested that rare clonotypes within the primary response had now come to dominate memory. Five additional A2. 01-positive IM patients were studied prospectively for Vbeta subset representation within primary and memory epitope-specific populations as identified by tetramer staining. In each case, the primary response contained large Vbeta2, Vbeta16, or Vbeta22 components, and in three of five cases the originally dominant Vbeta was represented very poorly, if at all, in memory. We conclude 1) that an EBV epitope-specific primary response large enough to account for up to 10% CD8(+) T cells in IM blood may nevertheless be dominated by just a few highly expanded clonotypes, and 2) that with persistent viral challenge such dominant T cell clonotypes may be lost and replaced by others in memory.     

Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype

We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4(+) and CD8(+) T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8(+). The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8(+) T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool.     

A Higher Activation Threshold of Memory CD8+ T Cells Has a Fitness Cost That Is Modified by TCR Affinity during Tuberculosis

T cell vaccines against Mycobacterium tuberculosis (Mtb) and other pathogens are based on the principle that memory T cells rapidly generate effector responses upon challenge, leading to pathogen clearance. Despite eliciting a robust memory CD8⁺ T cell response to the immunodominant Mtb antigen TB10.4 (EsxH), we find the increased frequency of TB10.4-specific CD8⁺ T cells conferred by vaccination to be short-lived after Mtb challenge. To compare memory and naïve CD8⁺ T cell function during their response to Mtb, we track their expansions using TB10.4-specific retrogenic CD8⁺ T cells. We find that the primary (naïve) response outnumbers the secondary (memory) response during Mtb challenge, an effect moderated by increased TCR affinity. To determine whether the expansion of polyclonal memory T cells is restrained following Mtb challenge, we used TCRβ deep sequencing to track TB10.4-specific CD8⁺ T cells after vaccination and subsequent challenge in intact mice. Successful memory T cells, defined by their clonal expansion after Mtb challenge, express similar CDR3β sequences suggesting TCR selection by antigen. Thus, both TCR-dependent and -independent factors affect the fitness of memory CD8⁺ responses. The impaired expansion of the majority of memory T cell clonotypes may explain why some TB vaccines have not provided better protection.     

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.     

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.4_4-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.4_4-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.     

Selection of CD8+ T cells with highly focused specificity during viral persistence in the central nervous system

The relationships between T cell populations during primary viral infection and persistence are poorly understood. Mice infected with the neurotropic JHMV strain of mouse hepatitis virus mount potent regional CTL responses that effectively reduce infectious virus; nevertheless, viral RNA persists in the central nervous system (CNS). To evaluate whether persistence influences Ag-specific CD8+ T cells, functional TCR diversity was studied in spleen and CNS-derived CTL populations based on differential recognition of variant peptides for the dominant nucleocapsid epitope. Increased specificity of peripheral CTL from persistently infected mice for the index epitope compared with immunized mice suggested T cell selection during persistence. This was confirmed with CD8+ T cell clones derived from the CNS of either acutely (CTLac) or persistently (CTLper) infected mice. Whereas CTLac clones recognized a broad diversity of amino acid substitutions, CTLper clones exhibited exquisite specificity for the wild-type sequence. Highly focused specificity was CD8 independent but correlated with longer complementarity-determining regions 3 characteristic of CTLper clonotypes despite limited TCR alpha/beta-chain heterogeneity. Direct ex vivo analysis of CNS-derived mononuclear cells by IFN-gamma enzyme-linked immunospot assay confirmed the selection of T cells with narrow Ag specificity during persistence at the population level. These data suggest that broadly reactive CTL during primary infection are capable of controlling potentially emerging mutations. By contrast, the predominance of CD8+ T cells with dramatically focused specificity during persistence at the site of infection and in the periphery supports selective pressure driven by persisting Ag.     

Despite biased TRBV gene usage against a dominant HLA B57-restricted epitope, TCR diversity can provide recognition of circulating epitope variants

The role of epitope-specific TCR repertoire diversity in the control of HIV-1 viremia is unknown. Further analysis at the clonotype level is important for understanding the structural aspects of the HIV-1 specific repertoire that directly relate to CTL function and ability to suppress viral replication. In this study, we performed in-depth analysis of T cell clonotypes directed against a dominantly recognized HLA B57-restricted epitope (KAFSPEVIPMF; KF11) and identified common usage of the TCR beta-chain TRBV7 in eight of nine HLA B57 subjects examined, regardless of HLA B57 subtype. Despite this convergent TCR gene usage, structural and functional assays demonstrated no substantial difference in functional or structural avidity between TRBV7 and non-TRBV7 clonotypes and this epitopic peptide. In a subject where TRBV7-usage did not confer cross-reactivity against the dominant autologous sequence variant, another circulating TCR clonotype was able to preferentially recognize the variant peptide. These data demonstrate that despite selective recruitment of TCR for a conserved epitope over the course of chronic HIV-1 infection, TCR repertoire diversity may benefit the host through the ability to recognize circulating epitope variants.     

The repertoire of CD4+ CD28- T cells in rheumatoid arthritis.

BACKGROUND: While oligoclonality of circulating CD4- CD8 and of CD8+ T cells is not uncommon, clonal dominance within the CD4 compartment is not frequently found in healthy individuals. In contrast, the majority of patients with rheumatoid arthritis (RA) have clonally expanded CD4+ T cell populations. Previous studies have demonstrated that these clonogenic CD4+ T cells do not express the CD28 molecule. To examine the correlation between CD28 expression and clonal proliferation, we have analyzed the T cell receptor (TCR) diversity of CD4+ CD28- T cells in normal individuals and in RA patients. MATERIAL AND METHODS: The size of the peripheral blood CD4+ CD28- compartment was determined in 30 healthy individuals and 30 RA patients by two-color FACS analysis. In 10 RA patients and five controls with more than 2.5% CD4+ CD28- T cells, TCR BV gene segment usage was analyzed with 19 BV-specific antibodies. Oligoclonality was assessed in sorted CD4+ CD28+ and CD28- T cells using TCR BV-BC-specific polymerase chain reaction and size fractionation. Clonal dominance was confirmed by direct sequencing. RESULTS: The CD4+ CD28- T cell compartment was expanded to more than 2.5% in 70% of the RA patients and 30% of the normal individuals. Compared with the CD4+ CD28+ T cells, the TCR BV gene segment usage among CD4+ CD28- cells was grossly skewed with the dominance of single BV elements. Molecular TCR analysis provided evidence for oligoclonality in 17 of 21 expanded BV elements. In two unrelated RA patients who shared both HLA-DRB1 alleles, the TCR beta-chain sequences of dominant clonotypes were highly conserved. CONCLUSIONS: Oligoclonality is a characteristic feature of CD4+ CD28- T cells which are expanded in some healthy individuals and in the majority of RA patients. The lack of CD28 expression is a common denominator of CD4+, CD8+, and CD4- CD8- T cells prone to develop clonal dominance. The limited TCR diversity of clonal CD4+ CD28- populations in RA patients suggests that these T cells recognize a limited spectrum of antigens. The fact that the majority of individuals with marked expansions and oligoclonality of CD4+ CD28- T cells are RA patients suggests a role for these unusual lymphocytes in the pathogenetic events leading to RA.     

Peroxiredoxin II Regulates Effector and Secondary Memory CD8+ T Cell Responses

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H₂O₂ on an antigen-specific CD8⁺ T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8⁺ T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8⁺ T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8⁺ T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8⁺ T cell pool. During chronic viral infection, increased antigen-specific CD8⁺ T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8⁺ T cell expansion, secondary memory generation, and immunopathology.     

Characterization of T-cell repertoire in hairy cell leukemia patients before and after recombinant immunotoxin BL22 therapy

We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.     

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates

Antigen-specific CD8⁺ T cells play a key role in the host’s antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8⁺ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8⁺ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8⁺ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8⁺ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.     

A TCR Affinity Threshold Regulates Memory CD4 T Cell Differentiation following Vaccination

Diverse Ag-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein Ags with TLR agonists are very potent at inducing T cell immune responses, but their capacity to elicit stable and diverse memory CD4 T cell repertoires has not been evaluated. In this study, we examined the evolution of a complex Ag-specific population during the transition from primary effectors to memory T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires, but peptide vaccines skewed the memory CD4 TCR repertoire toward high-affinity clonotypes whereas protein vaccines maintained low-affinity clonotypes in the memory compartment. CD27-mediated signaling was essential for the maintenance of low-affinity clonotypes after protein vaccination but was not sufficient to promote their survival following peptide vaccination. The rapid culling of the TCR repertoire in peptide-immunized mice coincided with a prolonged proliferation phase during which low-affinity clonotypes disappeared despite exhibiting no sign of enhanced apoptosis. Our study reveals a novel affinity threshold for memory CD4 T cell differentiation following vaccination and suggests a role for nonapoptotic cell death in the regulation of CD4 T cell clonal selection.     

Antigen-specific CD4 effector T cells: Analysis of factors regulating clonal expansion and cytokine production: Clonal expansion and cytokine production by CD4 effector T cells

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4⁺ antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4⁺ T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRβ crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high “avidity” effector and memory T cells in response to pathogen are discussed.     

Selection and long-term persistence of reactive CTL clones during an EBV chronic response are determined by avidity, CD8 variable contribution compensating for differences in TCR affinities.

Recent studies have suggested that the diversity of TCR repertoire after primary immunization is conserved in memory T cells and that a progressive narrowing of this repertoire may take place during recall infections. It now remains to be investigated which parameters determine the repertoire of the memory response and possibly restrict its diversity after subsequent antigenic challenges. To address this question, we took advantage of a panel of CD8+ T cell clones from the joint of a rheumatoid arthritis patient and selected for their reactivity against a single MHC/peptide complex. Characterization of both TCR chains documented a great diversity among those clones and the persistence of clonotypes over a 2-yr period. Strikingly, despite the observed repertoire heterogeneity, all clones displayed a narrow range of MHC/peptide density requirements in cytotoxicity assays (ED50 between 9 and 36 nM). TCR affinities were then indirectly estimated by blocking CD8 interaction with an anti-CD8 mAb. We found a wide range of TCR affinities among the different clonotypes that segregated with Vbeta usage. We thus propose that during an in vivo chronic response, a narrow range of avidity of the TCR-CD8 complex conditions long-term clonotype persistence, and that the level of CD8 contribution is adjusted to keep clonotypes with variable TCR affinities within this avidity window.     

Epstein-Barr Virus Infection of Na?ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD⁺ CD27⁺ non-switched memory (NSM) and IgD⁻ CD27⁺ switched memory (SM) B cells, not in IgD⁺ CD27⁻ naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL''s evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.