Conceived and designed the experiments: GB JM UB. Performed the experiments: DA. Analyzed the data: JM DA UB. Contributed reagents/materials/analysis tools: GB HB AP SB AM KW. Wrote the paper: JM DA UB.
The authors have declared that no competing interests exist.
Epstein-Barr virus (EBV) is associated with a number of human malignancies. EBV-positive post-transplant lymphoproliferative disease in solid organ and hematopoietic stem cell transplant recipients has been successfully treated by the adoptive transfer of polyclonal EBV-specific T cell lines containing CD4+ and CD8+ T cell components. Although patients receiving T cell preparations with a higher CD4+ T cell proportion show better clinical responses, the specificity of the infused CD4+ component has remained completely unknown.
We generated LCL-stimulated T cell lines from 21 donors according to clinical protocols, and analyzed the antigen specificity of the CD4+ component in EBV-specific T cell preparations using a genetically engineered EBV mutant that is unable to enter the lytic cycle, and recombinantly expressed and purified EBV proteins. Surprisingly, CD4+ T cell lines from acutely and persistently EBV-infected donors consistently responded against EBV lytic cycle antigens and autoantigens, but barely against latent cycle antigens of EBV hitherto considered principal immunotherapeutic targets. Lytic cycle antigens were predominantly derived from structural proteins of the virus presented on MHC II via receptor-mediated uptake of released viral particles, but also included abundant infected cell proteins whose presentation involved intercellular protein transfer. Importantly, presentation of virion antigens was severely impaired by acyclovir treatment of stimulator cells, as currently performed in most clinical protocols.
These results indicate that structural antigens of EBV are the immunodominant targets of CD4+ T cells in LCL-stimulated T cell preparations. These findings add to our understanding of the immune response against this human tumor-virus and have important implications for the improvement of immunotherapeutic strategies against EBV.
Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus implicated in the etiology of several tumors of lymphoid and epithelial origin
In vivo, outgrowth of latently infected growth-transformed B cells is curtailed by T cells. The importance of T cell-mediated immune responses in maintaining asymptomatic viral persistence is emphasized by the clinical observation that patients with T cell dysfunction are at risk of developing life-threatening EBV-associated lymphoproliferative disease
The proven safety and efficacy of adoptive T cell therapy for PTLD in HSCT recipients has provided an important proof of principle for this form of immunotherapy, but owing to the considerable technical requirements and financial implications of extensive in vitro T cell culture, adoptive T cell therapy still has a limited role in the management of virus-associated complications in transplant patients
Moreover, the successful treatment of PTLD in immunocompromised transplant recipients has encouraged the extension of these protocols to treat EBV-associated tumors developing in the presence of an apparently competent immune system, e.g. nasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD). First clinical experience indicates that LCL-stimulated T cell lines may cause tumor regression in some cases but clinical responses are often partial and transient
To increase clinical efficacy of the T cell preparations and to implement this treatment modality as a conventional therapeutic option, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. Prerequisite for the realization of these objectives is the knowledge of the relevant T cell antigens. Here, we studied the specificity of the CD4+ T cell component in LCL-stimulated T cell preparations.
Studies on material of human origin were approved by the ethics committees of the universities involved, and informed consent was obtained from all donors or their guardians. Blood samples from serologically confirmed cases of acute IM were obtained from the Children's Hospital, Munich University of Technology. Cord blood samples were provided by the University Hospital of the Ludwig Maximilians University, Munich. Mononuclear cells were isolated from blood samples by density gradient centrifugation on Ficoll-Paque (GE Healthcare). All donors were HLA-typed using PCR-based methods.
LCL and mini-LCL were established by infection of primary B cells with B95.8 virus and the genetically engineered mini-EBV strain, respectively
T cell lines were established by LCL or mini-LCL stimulation as in clinical protocols
Dendritic cells were differentiated from precursors in peripheral blood as described
For FACS analysis of T cells, FITC or PE-conjugated monoclonal antibodies against human CD4, CD8, and TCRα/β were used (all from Becton-Dickinson). TCR-Vβ usage by T cells was analyzed by RT-PCR and Southern blot. cDNA was synthesized from total RNA extracted from T cells and PCR performed using primers specific for the variable regions of the different human TCR-Vβ chains
Cell free supernatant from B95.8 cells was filtered through a 0.8 µm filter and ultracentrifuged at 25,000× g for 3 hours in a SW28 rotor (Beckman Coulter). The supernatant was removed and the virus rich pellet resuspended in 1/20 volume of the original culture supernatant. The number of EBV genome equivalents (geq)/ml of this virus concentrate was determined by semi-quantitative real-time PCR using primers directed to the BALF5 gene
The following EBV proteins were selected: the latent proteins EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, LMP1, LMP2A; the immediate early lytic cycle proteins BZLF1 and BRLF1, the early lytic cycle proteins BALF1, BALF2, BALF3, BALF5, BaRF1, BARF1, BBLF2/BBLF3, BBLF4, BDLF4, BFLF2, BFRF1, BGLF3, BGLF5, BHRF1, BKRF3, BKRF4, BLLF3, BMLF1, BMRF1, BORF2, BRRF1, BVRF2, BXLF1, and the late lytic cycle proteins BALF4, BBRF1, BBRF2, BBRF3, BcLF1, BcRF1, BCRF1, BDLF1, BDLF3, BFRF3, BGLF1, BGLF2, BILF2, BKRF2, BLLF1, BLRF1, BLRF2, BNRF1, BOLF1, BORF1, BSLF1, BSRF1, BXLF2, BXRF1, BZLF2. The cDNAs coding for latent cycle proteins were kindly provided by Dr. W. Hammerschmidt (GSF, Munich), or cloned from latently infected cells. The lytic cycle genes were amplified by PCR from B95.8 virus DNA and all genes cloned into the CMV promoter/enhancer driven mammalian expression vector pCMV-EHis, tagging the EBV genes at their 3′ end with sequences coding for the epitope recognized by the monoclonal α-EBNA1 antibody 1H4, and a His-tag consisting of six consecutive histidines.
For recombinant protein expression, the plasmids were transiently transfected into HEK293T cells using the calcium phosphate transfection method
Using autologous LCL as stimulators, T cell lines were established from mononuclear cells of umbilical or peripheral blood of 21 individuals; five cord blood donors, eight patients with IM, and eight healthy adult volunteers of whom seven were EBV-seropositive and one EBV-seronegative. As described for LCL-stimulated T cell lines prepared for clinical applications
T cell lines established from EBV-positive donors by LCL stimulation lysed autologous LCL but not PHA blasts after 4–8 passages at different effector-to-target (E:T) ratios. (B) FACS analysis of CD4+ cell lines established from LCL-stimulated bulk T cell lines by magnetic sorting demonstrated that >95% of the cells were TCRα/β+ and CD4+. (C) As demonstrated for donor GB, all TH cell lines established from healthy virus carriers responded against autologous and MHCII-matched allogeneic LCL, as well as MHCII-matched EBV-positive (BL41-B95.8) but not EBV-negative (BL41) Burkitt's lymphoma cell lines. TH cell lines from IM patients showed similar responses against autologous LCL, but as exemplified by the T cell line from IM4, some of these lines also recognized EBV-negative BL cell lines. (D) LCL-stimulated TH cell lines from EBV-negative donors showed minimal if any responses against autologous LCL, but vigorous responses against some allogeneic targets. (E) EBV-reactive TH cell lines secreted GM-CSF, IFN-γ, and TNF-α, but not IL-4, IL-10, IL-17, or TGF-β1 in response to stimulation with autologous (GB) or MHCII-matched allogeneic (JM) LCL, or non-specific activation by PHA. The MHC-mismatched LCL DA served as negative control. The following standards were included: GM-CSF: 1,900 pg/ml; IFN-γ: pg/ml; IL-4: 250 pg/ml; IL-10: 2,100 pg/ml; TNF-α: 2,900 pg/ml; TGF-β1: 1,450 pg/ml; IL-17: 1,700 pg/ml. (F) The T cell line IM7 displayed a novel “non-responder” phenotype. This T cell line proliferated in response to stimulation with autologous LCL and IL-2, but failed to secrete any of the indicated cytokines even after stimulation with autologous LCL plus PHA.
The five T cell lines derived from cord blood, and the T cell line from the EBV-seronegative healthy adult barely recognized autologous LCL (
Upon target cell recognition all LCL-reactive T cell lines predominantly secreted Th1 cytokines (
The exclusive recognition of EBV-positive but not EBV-negative targets by the T cell lines established from all healthy virus carriers and three IM patients suggested that these T cells were directed against latent cycle proteins of EBV. To define the TH cell antigens molecularly, all eight antigenically distinct latent cycle proteins of EBV were recombinantly expressed and the purified proteins pulsed on autologous PBMC, which were subsequently used as targets for the EBV-specific T cells. Efficient presentation of peptides derived from latent cycle proteins on MHC II was verified in control experiments using CD4+ T cell clones specific for five of the latent antigens of EBV (data not shown). Surprisingly, except for two T cell lines showing weak responses against EBNA3C, none of the EBV-specific T cell lines recognized any of the latent cycle antigens of EBV (
EBV-specific TH cell lines from different donors at different passages were tested for recognition of autologous PBMC pulsed separately with the eight antigenically distinct latent cycle proteins of the virus. Except for the T cell lines from donors DA and MS, which showed weak responses against EBNA3C, neither early nor late passage TH cell lines responded against EBV latent cycle proteins.
To address whether LCL-stimulated T cells recognize lytic cycle antigens of EBV, mini-LCL incapable of expressing lytic cycle proteins were established by infecting B cells with a genetically engineered mutant strain of EBV and used as T cell targets
LCL-stimulated TH cell lines showing EBV reactivity were tested for recognition of autologous LCL and mini-LCL established by infection of B cells with an EBV mutant unable to enter the lytic cycle. After three to twenty passages, mini-LCL reactivity of all TH cell lines had dropped to background levels while responses against LCL were maintained even after extended periods of in vitro culture. (B) Responses against LCL and mini-LCL of different passage TH cell lines were assessed by IFN-γ ELISPOT. With the exception of the T cell line from SM, early passage TH cell lines from healthy virus carriers recognized LCL and mini-LCL, but responses against mini-LCL disappeared with further rounds of stimulation. By contrast, early and late passage T cell lines from IM3, which had failed to show EBV-reactivity in earlier experiments, responded similarly against both types of target cells. SFC, spot forming cells.
The weak and transient responses against EBNA3C detected in two of the T cell lines established from healthy virus carriers implied that T cells specific for latent cycle antigens expanded under these in vitro culture condition, albeit less efficiently than lytic cycle antigen-specific TH cells. To assess whether latent cycle antigen-specific T cells are a subdominant component of the LCL-stimulated TH cell response, CD4+ PBMC from the donors DA and JM were repeatedly stimulated with autologous mini-LCL. These donors were chosen because TH cell lines and clones specific for EBV latent cycle antigens had been established previously from their peripheral blood, predicating the presence of such TH cell specificities in the peripheral memory compartment (data not shown). The resulting T cell lines responded similarly against autologous mini-LCL and LCL. Surprisingly, except for weak responses against EBNA3C in donor DA, these lines failed to recognize autologous PBMC or DC pulsed with any of the latent cycle antigens of EBV even after more than 25 passages, demonstrating that these T cell were not specific for latent antigens of EBV, but targeted cellular antigen(s) (
To investigate if the LCL but not mini-LCL-reactive TH cell lines recognized lytic cycle antigens, we cloned 50 of the more than 80 different lytic cycle genes of EBV including the immediate early antigens
The antigens recognized by TH cell lines that responded against LCL but had lost mini-LCL reactivity were identified using mini-LCL pulsed with recombinant EBV proteins. Responses of three representative CD4+ T cell lines (IM1, DA, and GB) against 30 different lytic cycle proteins are shown.
Donor | dominant antigens | subdominant antigens |
IM1 | BcLF1 | BFRF3, BXLF2 |
IM2 | BXLF2 | BDLF1, BNRF1 |
IM5 | BNRF1 | BALF2 |
GB | BNRF1 | BXRF1, BORF1, BDLF1, BBRF3 |
JM | BALF2 | BDLF1, BXRF1, BALF4 |
DA | BVRF2 | BNRF1, BCRF1, BORF1, EBNA3C |
MS | BALF4 | EBNA3C |
SM | BALF2, BNRF1 | BMRF1 |
MA | BMRF1, BNRF1 | Nd |
TK | BORF1 | Nd |
The antigens recognized by the EBV-reactive TH cell lines were identified by using PBMC or mini-LCL pulsed with single latent or lytic cycle proteins of EBV as targets. Responses against dominant antigens were maintained up to fifty restimulations, while responses against subdominant antigens were detected at early passages of the TH cell lines only. Nd, not determined.
Although late passage T cell lines usually responded against a single lytic cycle antigen, these experiments left unresolved whether these lines were still oligoclonal and contained additional specificities that remained undetected in these experiments e.g. lytic cycle antigens that had not been included in this study. To address this issue, two sets of experiments were performed. First, selected T cell lines were cloned by limiting dilution and analyzed for antigen specificity by assessing recognition of LCL, mini-LCL, and mini-LCL pulsed with proteins identified as targets of the parental T cell line. This analysis revealed that only a portion of the single cell outgrowths was of expected specificity. For example, the clones obtained from the T cell line MA passage p17 could be subdivided into four groups: (i) those that recognized BNRF1 or BMRF1, the previously identified targets of the parental T cell line, (ii) those that recognized LCL but neither mini-LCL alone nor mini-LCL pulsed with the recombinant BNRF1 or BMRF1, (iii) those that responded against LCL as well as mini-LCL and (iv) those that secreted neither GM-CSF nor IFN-γ upon co-culture with the target cells (
Given the low percentage of usually less than 1% of cells in an LCL culture that spontaneously become permissive for lytic replication, it was surprising to find that lytic cycle proteins of EBV are the immunodominant targets recognized by LCL-stimulated T cell lines. To investigate if structural proteins of EBV are presented via the receptor-mediated presentation pathway recently described for EBV glycoproteins
CD4+ T cells specific for BLLF1, BMRF1, BcLF1 or BNRF1 were tested for recognition of mini-LCL pulsed with purified viral particles. Whereas BcLF1, BLLF1, and BNRF1-specific T cells responded against mini-LCL pulsed with less than 1 genome equivalent (geq) of the virus/cell, BMRF1-specific T cells failed to recognize mini-LCL pulsed with much higher doses of virus. (B) To detect transfer of antigen between cells, BMRF1-specific T cells were tested for recognition of mini-LCL, MHC-mismatched LCL, and the mix of these two lines. While neither line alone was recognized by the T cells, 24 hours of co-culture sensitized the cell mix for recognition.
To preclude transfer of infectious virus into patients, T cell lines for clinical use are usually prepared by stimulation with acyclovir-treated LCL
LCL either left untreated or treated with acyclovir for two weeks were used as targets for BMRF1 (A), autoantigen (B), or BNRF1-specific T cells (C). Acyclovir treatment neither affected presentation of the autoantigen nor the EBV early lytic cycle antigen BMRF1, but severely reduced the presentation of the virion antigen BNRF1. (D) T cell lines generated by repeated stimulation of peripheral blood CD4+ cells with acyclovir-treated LCL recognized LCL and mini-LCL that had been pulsed with purified EBV particles, suggesting that late lytic cycle antigen-specific T cells still expand under these stimulation conditions.
The reconstitution of EBV-specific immunity in HSCT recipients by the adoptive transfer of polyclonal virus-specific T cell lines has provided an important proof of principle for immunotherapy of EBV-associated tumors, and for cancer immunotherapy in general
The second unexpected finding of this study was that LCL-stimulated TH cell lines contained a high proportion of autoreactive T cells, which either displayed a typical Th1, or a novel “non-responder” phenotype. The latter T cells were detected among T cell clones established from most of the lines and dominated the late passage T cell line from IM7, which makes them a relevant component of the LCL-stimulated TH cell population. The definition of T cell effector functions is essential for a more detailed characterization of this unusual T cell subset. Autoreactive T cells of Th1 type were detected in EBV-infected individuals only, and these specificities dominated the LCL-stimulated T cell cultures from several IM patients, suggesting a link between acute EBV infection and the induction of autoreactive TH cell responses. Of note, autoreactive T cells have recently been described as component of the CD4+ T cell response that suppresses the outgrowth of LCL from newly EBV-infected B cells in regression assays
The most important finding of this study was the unexpected immunodominance of lytic cycle antigens. Most of these antigens were derived from late lytic gene products that belong to the group of structural proteins of EBV. This immunodominance may be a reflection of the efficient presentation of virion antigens on MHC II following receptor-mediated uptake and processing in the lytic compartment
The identification of the immunodominant and subdominant antigens of LCL-stimulated TH cell preparations has several clinical implications. First, in order to minimize residual infectious viral particles within adoptively transferred T cells, most currently applied clinical protocols use acyclovir to suppress virus production in stimulator LCL
Although most EBV-associated tumors express MHC II, the low number of tumor cells undergoing lytic replication in vivo challenges the concept of an analogous role of virion-specific CD4+ T cells in tumor control. However, the efficient transfer of virion antigens to bystander cells by receptor-mediated uptake of released viral particles, which results in TH cell recognition of target cells incubated with less than one viral particle per cell, suggests that only few tumor cells undergoing lysis may sensitize a large proportion of tumor cells for TH cell recognition. Moreover, radio/chemotherapy of EBV-positive tumors in vivo is associated with the induction of lytic replication in a significant portion of tumor cells, and more selective compounds for reactivating EBV from latency are currently evaluated
Latent cycle proteins of EBV are not the dominant targets of mini-LCL stimulated TH cell lines. Mini-LCL-stimulated CD4+ T cell lines from donor DA and JM were tested for recognition of latent cycle proteins of EBV by using PBMC or DC preincubated for 24 hours with recombinant latent cycle proteins as targets in T cell cytokine secretion assays. The T cells from donor JM failed to show above-background response against any of the latent cycle proteins whereas the T cells from donor DA showed only minimal response against EBNA3C.
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Late passage LCL-stimulated CD4+ T cell lines are oligoclonal. Single cell clones of the LCL-stimulated T cell line from donor MA at passage 17 were tested for recognition of LCL, mini-LCL, and mini-LCL pulsed with BNRF1 or BMRF1, the antigens recognized by the parental T cell line. Of 16 clones analyzed, 5 were BMRF1-specific, 1 was BNRF1-specific, 4 recognized LCL but not mini-LCL, 2 showed significant responses against LCL and mini-LCL, and 4 failed to secrete IFN-γ in response to any of the target cells.
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T cell receptor Vβ chain analysis of late passage T cell lines. T cell receptor Vβ chain expression of TH cell lines, that had shown lytic cycle antigen specificity, was analyzed by RT-PCR using Vβ chain specific primers and subsequent Southern blot hybridization of the PCR products. Even late passage T cell lines still expressed more than one Vβ chain, demonstrating that the T cell lines were still oligoclonal. The TH cell line from donor SM, that had already lost mini-LCL reactivity after four stimulations, still expressed multiple Vβ chains, indicating that many different lytic cycle antigen-specific TH cells may exist in healthy virus carriers.
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We thank Brigitte Lechner and Heike Christoph for providing outstanding technical assistance.