A metabolic dependency of EBV can be targeted to hinder B cell transformation.

Following infection of B cells, Epstein Barr virus (EBV) engages host pathways that mediate cell proliferation and transformation, contributing to the propensity of the virus to drive immune dysregulation and lymphomagenesis. We found that the EBV protein EBNA2 initiates NAD de novo biosynthesis by driving expression of the metabolic enzyme IDO1 in infected B cells. Virus-enforced NAD production sustained mitochondrial complex I activity, to match ATP-production with bioenergetic requirements of proliferation and transformation. In transplant patients, IDO1 expression in EBV-infected B cells, and a serum signature of increased IDO1 activity, preceded development of lymphoma. In humanized mice infected with EBV, IDO1 inhibition reduced both viremia and lymphomagenesis. Virus-orchestrated NAD biosynthesis is, thus, a druggable metabolic vulnerability of EBV-driven B cell transformation-opening therapeutic possibilities for EBV-related diseases.

T Cells Directed against the Metastatic Driver Chondromodulin-1 in Ewing Sarcoma: Comparative Engineering with CRISPR/Cas9 vs. Retroviral Gene Transfer for Adoptive Transfer.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.

Association between IgG responses against the nucleocapsid proteins of alphacoronaviruses and COVID-19 severity.

Understanding immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to contain the COVID-19 pandemic. Using a multiplex approach, serum IgG responses against the whole SARS-CoV-2 proteome and the nucleocapsid proteins of endemic human coronaviruses (HCoVs) were measured in SARS-CoV-2-infected donors and healthy controls. COVID-19 severity strongly correlated with IgG responses against the nucleocapsid (N) of SARS-CoV-2 and possibly with the number of viral antigens targeted. Furthermore, a strong correlation between COVID-19 severity and serum responses against N of endemic alpha- but not betacoronaviruses was detected. This correlation was neither caused by cross-reactivity of antibodies, nor by a general boosting effect of SARS-CoV-2 infection on pre-existing humoral immunity. These findings raise the prospect of a potential disease progression marker for COVID-19 severity that allows for early stratification of infected individuals.

Revisiting IgG Antibody Reactivity to Epstein-Barr Virus in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Its Potential Application to Disease Diagnosis.

Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients.

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 (TCRß) 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.

Defining the RBPome of primary T helper cells to elucidate higher-order Roquin-mediated mRNA regulation.

Post-transcriptional gene regulation in T cells is dynamic and complex as targeted transcripts respond to various factors. This is evident for the Icos mRNA encoding an essential costimulatory receptor that is regulated by several RNA-binding proteins (RBP), including Roquin-1 and Roquin-2. Here, we identify a core RBPome of 798 mouse and 801 human T cell proteins by utilizing global RNA interactome capture (RNA-IC) and orthogonal organic phase separation (OOPS). The RBPome includes Stat1, Stat4 and Vav1 proteins suggesting unexpected functions for these transcription factors and signal transducers. Based on proximity to Roquin-1, we select ~50 RBPs for testing coregulation of Roquin-1/2 targets by induced expression in wild-type or Roquin-1/2-deficient T cells. Besides Roquin-independent contributions from Rbms1 and Cpeb4 we also show Roquin-1/2-dependent and target-specific coregulation of Icos by Celf1 and Igf2bp3. Connecting the cellular RBPome in a post-transcriptional context, we find contributions from multiple RBPs to the prototypic regulation of mRNA targets by individual trans-acting factors.

Automated, flow-based chemiluminescence microarray immunoassay for the rapid multiplex detection of IgG antibodies to SARS-CoV-2 in human serum and plasma (CoVRapid CL-MIA).

In the face of the COVID-19 pandemic, the need for rapid serological tests that allow multiplexing emerged, as antibody seropositivity can instruct about individual immunity after an infection with SARS-CoV-2 or after vaccination. As many commercial antibody tests are either time-consuming or tend to produce false negative or false positive results when only one antigen is considered, we developed an automated, flow-based chemiluminescence microarray immunoassay (CL-MIA) that allows for the detection of IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD), spike protein (S1 fragment), and nucleocapsid protein (N) in human serum and plasma in less than 8 min. The CoVRapid CL-MIA was tested with a set of 65 SARS-CoV-2 serology positive or negative samples, resulting in 100% diagnostic specificity and 100% diagnostic sensitivity, thus even outcompeting commercial tests run on the same sample set. Additionally, the prospect of future quantitative assessments (i.e., quantifying the level of antibodies) was demonstrated. Due to the fully automated process, the test can easily be operated in hospitals, medical practices, or vaccination centers, offering a valuable tool for COVID-19 serosurveillance. Graphical abstract.

Immunoinformatic Analysis Reveals Antigenic Heterogeneity of Epstein-Barr Virus Is Immune-Driven.

Whole genome sequencing of Epstein-Barr virus (EBV) isolates from around the world has uncovered pervasive strain heterogeneity, but the forces driving strain diversification and the impact on immune recognition remained largely unknown. Using a data mining approach, we analyzed more than 300 T-cell epitopes in 168 published EBV strains. Polymorphisms were detected in approximately 65% of all CD8+ and 80% of all CD4+ T-cell epitopes and these numbers further increased when epitope flanking regions were included. Polymorphisms in CD8+ T-cell epitopes often involved MHC anchor residues and resulted in changes of the amino acid subgroup, suggesting that only a limited number of conserved T-cell epitopes may represent generic target antigens against different viral strains. Although considered the prototypic EBV strain, the rather low degree of overlap with most other viral strains implied that B95.8 may not represent the ideal reference strain for T-cell epitopes. Instead, a combinatorial library of consensus epitopes may provide better targets for diagnostic and therapeutic purposes when the infecting strain is unknown. Polymorphisms were significantly enriched in epitope versus non-epitope protein sequences, implicating immune selection in driving strain diversification. Remarkably, CD4+ T-cell epitopes in EBNA2, EBNA-LP, and the EBNA3 family appeared to be under negative selection pressure, hinting towards a beneficial role of immune responses against these latency type III antigens in virus biology. These findings validate this immunoinformatics approach for providing novel insight into immune targets and the intricate relationship of host defense and virus evolution that may also pertain to other pathogens.

The Epstein-Barr Virus Major Tegument Protein BNRF1 Is a Common Target of Cytotoxic CD4+ T Cells.

Cellular immunotherapy is a proven approach against Epstein-Barr virus (EBV)-driven lymphoproliferation in recipients of hematopoietic stem cells. Extending the applicability and improving the response rates of such therapy demands improving the knowledge base. We studied 23 healthy donors for specific CD4+ T cell responses against the viral tegument protein BNRF1 and found such T cells in all seropositive donors, establishing BNRF1 as an important immune target in EBV. We identified 18 novel immune epitopes from BNRF1, all of them generated by natural processing of the full-length protein from virus-transformed lymphoblastoid cell lines (LCL). BNRF1-specific CD4+ T cells were measured directly ex vivo by a cytokine-based method, thus providing a tool to study the interaction between immunity and infection in health and disease. T cells of the cytotoxic Th1 type inhibited the proliferation of autologous LCL as well as virus-driven transformation. We infer that they are important in limiting reactivations to subclinical levels during health and reducing virus propagation during disease. The information obtained from this work will feed into data sets that are indispensable in the design of patient-tailored immunotherapeutic approaches, thereby enabling the stride toward broader application of T cell therapy and improving clinical response rates.IMPORTANCE Epstein-Barr virus is carried by most humans and can cause life-threatening diseases. Virus-specific T cells have been used in different clinical settings with variable success rates. One way to improve immunotherapy is to better suit T cell generation protocols to viral targets available in different diseases. BNRF1 is present in viral particles and therefore likely available as a target for T cells in diseases with virus amplification. Here, we studied healthy Epstein-Barr virus (EBV) carriers for BNRF1 immunogenicity and report our results indicating BNRF1 to be a dominant target of the EBV-specific CD4+ T cell response. BNRF1-specific CD4+ T cells were found to be cytotoxic and capable of limiting EBV-driven B cell transformation in vitro The findings of this work contribute to forwarding our understanding of host-virus interactions during health and disease and are expected to find direct application in the generation of specific T cells for immunotherapy.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma.

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Progress in EBV Vaccines.

The Epstein-Barr virus (EBV) is a ubiquitous pathogen that imparts a significant burden of disease on the human population. EBV is the primary cause of infectious mononucleosis and is etiologically linked to the development of numerous malignancies. In recent years, evidence has also been amassed that strongly implicate EBV in the development of several autoimmune diseases, including multiple sclerosis. Prophylactic and therapeutic vaccination has been touted as a possible means of preventing EBV infection and controlling EBV-associated diseases. However, despite several decades of research, no licensed EBV vaccine is available. The majority of EBV vaccination studies over the last two decades have focused on the major envelope protein gp350, culminating in a phase II clinical trial that showed soluble gp350 reduced the incidence of IM, although it was unable to protect against EBV infection. Recently, novel vaccine candidates with increased structural complexity and antigenic content have been developed. The ability of next generation vaccines to safeguard against B-cell and epithelial cell infection, as well as to target infected cells during all phases of infection, is likely to decrease the negative impact of EBV infection on the human population.

Antibodies conjugated with viral antigens elicit a cytotoxic T cell response against primary CLL ex vivo.

Chronic lymphocytic leukemia (CLL) is the most frequent B cell malignancy in Caucasian adults. The therapeutic armamentarium against this incurable disease has recently seen a tremendous expansion with the introduction of specific pathway inhibitors and innovative immunotherapy. However, none of these approaches is curative and devoid of side effects. We have used B-cell-specific antibodies conjugated with antigens (AgAbs) of the Epstein-Barr virus (EBV) to efficiently expand memory CD4+ cytotoxic T lymphocytes (CTLs) that recognized viral epitopes in 12 treatment-naive patients with CLL. The AgAbs carried fragments from the EBNA3C EBV protein that is recognized by the large majority of the population. All CLL cells pulsed with EBNA3C-AgAbs elicited EBV-specific T cell responses, although the intensity varied across the patient collective. Interestingly, a large proportion of the EBV-specific CD4+ T cells expressed granzyme B (GrB), perforin, and CD107a, and killed CLL cells loaded with EBV antigens with high efficiency in the large majority of patients. The encouraging results from this preclinical ex vivo study suggest that AgAbs have the potential to redirect immune responses toward CLL cells in a high percentage of patients in vivo and warrant the inception of clinical trials.

Immunogenic particles with a broad antigenic spectrum stimulate cytolytic T cells and offer increased protection against EBV infection ex vivo and in mice.

The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection.

Immunization with a recombinant fusion protein protects mice against Helicobacter pylori infection.

More than 50% of the world's population is infected with the bacterium Helicobacter pylori. If left untreated, infection with H. pylori can cause chronic gastritis and peptic ulcer disease, which may progress into gastric cancer. Owing to the limited efficacy of anti-H. pylori antibiotic therapy in clinical practice, the development of a protective vaccine to combat this pathogen has been a tempting goal for several years. In this study, a chimeric gene coding for the antigenic parts of H. pylori FliD, UreB, VacA, and CagL was generated and expressed in bacteria and the potential of the resulting fusion protein (rFUVL) to induce humoral and cellular immune responses and to provide protection against H. pylori infection was evaluated in mice. Three different immunization adjuvants were tested along with rFUVL: CpG oligodeoxynucleotides (CpG ODN), Addavax, and Cholera toxin subunit B. Compared to the control group that had received PBS, vaccinated mice showed significantly higher cellular recall responses and antigen-specific IgG2a, IgG1, and gastric IgA antibody titers. Importantly, rFUVL immunized mice exhibited a reduction of about three orders of magnitude in their stomach bacterial loads. Thus, adjuvanted rFUVL might be considered as a promising vaccine candidate for the control of H. pylori infection.

Clinical-grade generation of peptide-stimulated CMV/EBV-specific T cells from G-CSF mobilized stem cell grafts.

BACKGROUND: A major complication after allogeneic hematopoietic stem cell transplantation (aSCT) is the reactivation of herpesviruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Both viruses cause significant mortality and compromise quality of life after aSCT. Preventive transfer of virus-specific T cells can suppress reactivation by re-establishing functional antiviral immune responses in immunocompromised hosts. METHODS: We have developed a good manufacturing practice protocol to generate CMV/EBV-peptide-stimulated T cells from leukapheresis products of G-CSF mobilized and non-mobilized donors. Our procedure selectively expands virus-specific CD8+ und CD4+ T cells over 9 days using a generic pool of 34 CMV and EBV peptides that represent well-defined dominant T-cell epitopes with various HLA restrictions. For HLA class I, this set of peptides covers at least 80% of the European population. RESULTS: CMV/EBV-specific T cells were successfully expanded from leukapheresis material of both G-CSF mobilized and non-mobilized donors. The protocol allows administration shortly after stem cell transplantation (d30+), storage over liquid nitrogen for iterated applications, and protection of the stem cell donor by avoiding a second leukapheresis. CONCLUSION: Our protocol allows for rapid and cost-efficient production of T cells for early transfusion after aSCT as a preventive approach. It is currently evaluated in a phase I/IIa clinical trial.

Immunization with recombinant FliD confers protection against Helicobacter pylori infection in mice.

Nearly half of the world's population is infected with Helicobacter pylori. Clinical manifestations of this infection range from gastritis and peptic ulcers to gastric adenocarcinoma and lymphoma. Due to the emerging of antibiotic resistant strains and poor patient compliance of the antibiotic therapy, there is increasing interest in the development of a protective vaccine against H. pylori infection. The bacterial protein FliD forms a capping structure on the end of each flagellum which is critical to prevent depolymerization and structural degradation. In this study, the potential of FliD as a prospective H. pylori subunit vaccine was assessed. For this purpose, immunogenicity and protective efficacy of recombinant FliD (rFliD) from H. pylori was evaluated in C57BL/6 mice. Purified rFliD was formulated with different adjuvants and administered via subcutaneous or oral route. Subcutaneous immunization with rFliD elicited predominantly mixed Th1 and Th17 immune responses, with high titers of specific IgG1 and IgG2a. Splenocytes of immunized mice exhibited strong antigen-specific memory responses, resulting in the secretion of high amounts of IFN-γ and IL-17, and low levels of IL-4. Immunization with rFliD caused a significant reduction in H. pylori bacterial load relative to naïve control mice (p < 0.001), demonstrating a robust protective effect. Taken together, these results suggest that subcutaneous vaccination with rFliD formulated with CpG or Addavax could be considered as a potential candidate for the development of a subunit vaccine against H. pylori infection.

Epstein-Barr virus strain heterogeneity impairs human T-cell immunity.

The Epstein-Barr virus (EBV) establishes lifelong infections in > 90% of the human population. Although contained as asymptomatic infection by the immune system in most individuals, EBV is associated with the pathogenesis of approximately 1.5% of all cancers in humans. Some of these EBV-associated tumors have been successfully treated by the infusion of virus-specific T-cell lines. Recent sequence analyses of a large number of viral isolates suggested that distinct EBV strains have evolved in different parts of the world. Here, we assessed the impact of such sequence variations on EBV-specific T-cell immunity. With the exceptions of EBNA2 and the EBNA3 family of proteins, an overall low protein sequence disparity of about 1% was noted between Asian viral isolates, including the newly characterized M81 strain, and the prototypic EBV type 1 and type 2 strains. However, when T-cell epitopes including their flanking regions were compared, a substantial proportion was found to be polymorphic in different EBV strains. Importantly, CD4+ and CD8+ T-cell clones specific for viral epitopes from one strain often showed diminished recognition of the corresponding epitopes in other strains. In addition, T-cell recognition of a conserved epitope was affected by amino acid exchanges within the epitope flanking region. Moreover, the CD8+ T-cell response against polymorphic epitopes varied between donors and often ignored antigen variants. These results demonstrate that viral strain heterogeneity may impair antiviral T-cell immunity and suggest that immunotherapeutic approaches against EBV should preferably target broad sets of conserved epitopes including their flanking regions.

Epstein-Barr virus particles induce centrosome amplification and chromosomal instability.

Infections with Epstein-Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome.

Epstein-Barr viral miRNAs inhibit antiviral CD4+ T cell responses targeting IL-12 and peptide processing.

Epstein-Barr virus (EBV) is a tumor virus that establishes lifelong infection in most of humanity, despite eliciting strong and stable virus-specific immune responses. EBV encodes at least 44 miRNAs, most of them with unknown function. Here, we show that multiple EBV miRNAs modulate immune recognition of recently infected primary B cells, EBV's natural target cells. EBV miRNAs collectively and specifically suppress release of proinflammatory cytokines such as IL-12, repress differentiation of naive CD4(+) T cells to Th1 cells, interfere with peptide processing and presentation on HLA class II, and thus reduce activation of cytotoxic EBV-specific CD4(+) effector T cells and killing of infected B cells. Our findings identify a previously unknown viral strategy of immune evasion. By rapidly expressing multiple miRNAs, which are themselves nonimmunogenic, EBV counteracts recognition by CD4(+) T cells and establishes a program of reduced immunogenicity in recently infected B cells, allowing the virus to express viral proteins required for establishment of life-long infection.

Intrathecal CD8 T-cells of multiple sclerosis patients recognize lytic Epstein-Barr virus proteins.

BACKGROUND: The association between Epstein-Barr virus (EBV) and multiple sclerosis (MS) may involve intrathecal EBV-specific T-cell responses targeting the virus or indirectly, autoantigens. OBJECTIVE: Compare the prevalence and fine-specificity of EBV-specific T-cells in the cerebrospinal fluid (CSF) of patients with MS (n = 12), clinically-isolated syndrome (CIS) (n = 17) and other neurological diseases (OND) (n = 13). METHODS: Intrathecal EBV-specific T-cell reactivity was assayed using CSF-derived T-cell lines (CSF-TCL) and autologous EBV-transformed B-cells (autoBLCL) as antigen-presenting cells (APC). EBV proteins recognized by autoBLCL-specific CD8 T-cells were identified using human leukocyte antigen class I (HLA-I)-negative monkey cells as artificial APC, co-transfected with 59 different EBV genes and the corresponding patient's HLA-I alleles that were involved in autoBLCL T-cell reactivity. Reactivity towards the MS-associated autoantigen αB-crystallin (CRYAB) was determined analogously. RESULTS: CSF-TCL from CIS and MS patients had significantly higher frequencies of autoBLCL-reactive CD4 T-cells, compared to the OND patients. CIS patients also had significantly higher autoBLCL-reactive CD8 T cells, which correlated with reactive CD4 T-cell frequencies. AutoBLCL-specific CD8 T-cell responses of four CSF-TCL analyzed in detail were oligoclonal and directed to lytic EBV proteins, but not CRYAB endogenously expressed by autoBLCL. CONCLUSIONS: Enhanced intrathecal autoBLCL-specific T-cell reactivity, selectively directed towards lytic EBV proteins in two CSF-TCL, suggested a localized T-cell response to EBV in patients with MS. Our data warrant further characterization of the magnitude and breadth of intrathecal EBV-specific T-cell responses in larger patient cohorts.