Autologous EBV-transformed Lymphoblastoid Cell Lines Research Articles

Rapidly-Generated EBV-Specific T Cells (EBVST-cells) to Treat Type 2 Latency Lymphoma

Up to 30 % of Hodgkin and non-Hodgkin lymphomas carry the Epstein Barr virus (EBV) genome and express the viral latency type 2 proteins EBNA-1, LMP-1, LMP-2 and BARF-1 in a pattern termed Type 2 latency. We have previously shown that Epstein-Barr virus-specific T-cells (EBVSTs) can be expanded from the peripheral blood of lymphoma patients by stimulation with dendritic cells and autologous EBV-transformed lymphoblastoid cell lines (LCLs) modified with an adenoviral vector encoding LMP1 and LMP2. These EBVSTs induce complete clinical responses in over 50% of patients with active disease (Bollard et al J Clin Oncol 2014). Since the requirement for an LCL as an antigen-presenting cell added 3-4 months to the manufacturing time, we shortened and simplified the process. We replaced the LCL with autologous, activated T-cells together with HLA-negative K562 costimulatory cells as a source of antigen-presenting cell and replaced live virus (EBV) and adenoviral-vector components as a source of antigen with overlapping peptide libraries (pepmixes) spanning the all 4 Type 2 latency antigens (EBNA1, LMP1, LMP2 and BARF1). We also added IL4 and IL7, cytokines that increased the repertoire and expansion of EBVSTs as previously demonstrated by Gerdemann et al. (Mol Ther 2012) These changes reduced the manufacturing time to 3-4 weeks and increased the number of eligible patients, since patients with a short life expectancy were previously excluded and LCLs could not be generated from patients who had received the B-cell depleting antibody rituximab.However the majority of the first 14 EBVSTs generated from patients demonstrated low antigen-specificity. We hypothesized this poor response resulted from T-cell anergy and therefore replaced IL-4 with IL-15, which was reported to reverse tumor-specific anergy (Teague et al Nat Med 2006). This change increased the proliferative capacity and antigen specificity of patient EBVSTs by up to 10-fold, indicating a "rescue effect" of IL-15 on tolerant or anergized T-cells, and correlated with increased in vitro cytotoxicity against EBV antigen-positive target cells from 5% to 66%(n=5).In a Phase 1 clinical trial, we have infused 2 doses of EBVSTs generated with IL7 and IL15 into 24 patients with multiply-relapsed, EBV-positive Hodgkin or non-Hodgkin lymphoma, either as adjuvant therapy (after stem cell transplantation or chemotherapy (n=17) or as treatment for active disease (n=7). There were no immediate or delayed adverse events attributed to the cell infusion. Of 17 patients who received EBVSTs as adjuvant therapy, 15 remain in remission with follow up of 2 to 28 months while 2 subsequently progressed. Of 7 patients with active disease who received EBVSTs, two patients had complete responses (CRs) (sustained for 24+ and 18+ months so far), two had partial responses (PRs), one had stable disease and two patients progressed. The antitumor activity we observed was associated with increases not only in circulating EBVSTs in 14 of 22 patients followed for 3 months or more, but also in T-cells specific for one or more of the non-viral tumor associated antigens, MAGE-A4, SSX2, survivin, NY-ESO1 or PRAME. This antigen spreading was seen in 13 of the 14 patients with increased EBVSTs. In an effort to enhance T-cell mediated antitumor effects, we plan to incorporate PD-1 inhibition into the therapy for patients who have had a PR to this therapy, and evaluate lymphodepletion.This cell therapy product is also being tested in a multicenter Phase II trial (CITADEL, Cell Medica, NCT01948180), being run in the European Union, South Korea and the United States, in which patients with EBV positive NK-T lymphoma receive autologous EBVSTs grown with IL7 and IL15. Albeit early in the study, preliminary indications of activity have been observed. Of 2 evaluable patients with active disease at baseline, one had a PR at the 8 week follow up scan, based on independent radiological assessment, and of 2 patients with non-measurable disease at baseline, one remains in remission at 9+ month follow up. In addition, increases in circulating EBVSTs have been observed in 6 of 7 patients (minimum 2 week follow up). DisclosuresBollard:Cell Medica: Patents & Royalties. Inman:Cell Medica: Employment. Hodgkin:Cell Medica: Employment. Gunter:Cell Medica: Employment. Brenner:Viracyte: Equity Ownership; Cell Medica: Patents & Royalties. Heslop:Cell Medica: Patents & Royalties; Viracyte: Equity Ownership. Rooney:Viracyte: Equity Ownership; Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

EBV-Specific T-Cell Immunity in Pediatric Solid Organ Graft Recipients With Posttransplantation Lymphoproliferative Disease

Posttransplantation lymphoproliferative disease (PTLD) is an often Epstein-Barr virus (EBV)-associated mainly malignant complication after transplantation. We present data on EBV-specific T cells in children treated with rituximab with or without chemotherapy on the pediatric PTLD Pilot 2005 protocol. Peripheral blood mononuclear cells were isolated from 16 pediatric patients with PTLD, 4 transplanted children with EBV reactivation, and 18 healthy controls. EBV-specific T cells were quantified by flow cytometric detection of intracellular interferon-γ after stimulation with autologous EBV-transformed lymphoblastoid cell lines and correlated with EBV load in peripheral blood. At diagnosis, PTLD patients had similar numbers of EBV-specific CD4 and CD8 T cells as healthy EBV-positive controls. EBV-specific T cells tended to be lower in early PTLD compared with late PTLD. During treatment with rituximab, CD4 and/or CD8 EBV-specific T cells increased in most patients, possibly reflecting restored immunocompetence due to a reduction of immunosuppression as well as antigenic stimulation by cross-presentation of EBV antigen from destroyed B cells. However, this increase did not predict response to rituximab or chemotherapy. EBV load and circulating B cells became undetectable in most patients during rituximab therapy. B-cell recovery after treatment was accompanied by redetection of EBV in peripheral blood, which was controlled by T-cell responses in 11 of 11 evaluable cases. In pediatric PTLD patients, pretreatment EBV-specific T-cell numbers are in the range of healthy controls. These cells increased on reduction of immunosuppression and treatment with rituximab. Recurrence of EBV viremia during complete remission is matched by strong T-cell responses.

Longitudinal analysis of frequency and reactivity of epstein–barr virus‐specific T lymphocytes and their association with intermittent viral reactivation

Persistent Epstein-Barr virus (EBV) infection is controlled tightly by virus-specific T cells. EBV infection is reactivated intermittently over time, even in apparently healthy carriers. Changes in frequency and reactivity of memory T cells, particularly of CD8(+) origin, have not been assessed in this context. It is hypothesized that viral reactivation is facilitated by diminished EBV-specific T-cell immunity. To this end, blood samples from 14 healthy donors were collected at irregular time intervals for a period of about 1 year. Samples were screened for both EBV plasma viremia and increases in viral load in PBMCs as parameters of EBV reactivation. PBMCs were subject to IFN-γ ELISPOT analysis using the autologous EBV-transformed lymphoblastoid cell line (EBV-LCL) or appropriate HLA class I-restricted EBV peptides as stimulators. Frequencies of epitope-specific CD8(+) T cells were monitored further using HLA tetramers and flow cytometry. Twelve of 14 donors exhibited signs of asymptomatic EBV reactivation. Viral reactivation was accompanied by either substantially decreased IFN-γ responses against autologous EBV-LCL (eight of 12 study participants) and/or increased responses against particular EBV peptides (six of 12 donors). In seven persons with HLA-A2 and/or -B8 alleles numbers of HLA tetramer-positive CD8(+) T cells also varied over time, but showed no correlation to episodes of detectable viral activity. In summary, IFN-γ reactivity of EBV-specific T cells is not constant. Viral reactivation is detected preferably at times of diminished EBV-LCL-specific cellular immunity. However, increased reactivity of single immunodominant CD8(+) EBV-specific T-cell clones may occur in response to virus replication.

Repertoire and frequency of immune cells reactive to Epstein-Barr virus–derived autologous lymphoblastoid cell lines

AbstractAnswers to questions about frequency and repertoire of immune cells, relative contributions made by different types of immune cells toward the total Epstein-Barr virus (EBV)–directed response and the variation of such responses in healthy persons have been elusive because of disparities in assays, antigen presenting cells, and antigenic sources used in previous experiments. In this study, we addressed these questions using an assay that allowed direct comparison of responses generated by different types of cells of the immune system. This short-term (20-hour) ex vivo assay measured interferon-γ production by blood cells in response to autologous EBV-transformed lymphoblastoid cell lines (LCLs). Our experiments defined the variation in responses among persons and clearly distinguished 10 healthy EBV-immune from 10 healthy EBV-naive persons. In EBV-immune persons, 33% of responding cells were CD4+, 43.3% were CD8+, and 12.9% were γ-δ T cells. LCL-reactive CD8+ T cells were only 1.7-fold more frequent than similarly reactive CD4+T cells. Responses by γ-δ T cells were 6-fold higher in seropositive than in seronegative persons. Our findings emphasize the importance of CD4+ and γ-δ T-cell responses and have implications for immunotherapy and for identifying defects in T-cell populations that might predispose to development of EBV-associated lymphomas.

Efficient Generation of Tumor-Specific, Cytotoxic T Cells by Genetic Transfer of allo-MHC.

Immune responses to established cancer are difficult to evoke by vaccination of patients due to various mechanisms of tolerance. In contrast, T cells in allogeneic stem cell grafts can contribute to cure of a number of hematological malignancies by the graft-versus-leukemia (GvL) effect. However, unknown T cell specificities causing life-threatening graft-versus-host disease (GvHD) limit the applicability of this treatment. Selection of GvL activity would increase the efficiency, safety and applicability of allogeneic stem cell transplantation. Here, we describe a novel method for overcoming tolerance by generation of allo-restricted, antigen-specific T cells. These cells specifically recognize and kill melanoma tumor cells presenting the melanoma-associated MART-1 in context of the prevalent major histocompatibility complex (MHC) class I molecule HLA-A*0201. HLA-A*0201-negative peripheral blood mononuclear cells were co-cultured with autologous, monocyte-derived dendritic cells transfected with HLA-A*0201 mRNA and subsequently pulsed with the peptide ELAGIGILTV from the melanoma-associated antigen MART-1. Antigen-specific T cells were detected in 8/10 donors on day 12 using peptide-MHC-pentamers (median 0.28% of CD8+ T cells, range 0-4.17%), and in all donors by day 19. When needed, cells were restimulated on day 12 with HLA-A*0201 mRNA-transfected and peptide pulsed autologous EBV-transformed lymphoblastoid cell lines (EBV-LCL). MART-1 pentamer+ T cells from five donors were sorted and expanded. The cytotoxic T cell lines (CTLs) responded with production of interferon-γ, degranulation and lysis of target cells when stimulated with peptide-pulsed EBV-LCL, even at low peptide concentrations (10−5 M). Similar responses were seen to melanoma cell lines expressing HLA-A*0201 and MART-1 (FM-57, Malme3M). In contrast, MART-1+ melanoma cells lacking HLA-A*0201 (Mel202) induced responses only when transfected with HLA-A*0201. Starting out with 0.5×106 pentamer+ T cells, a potential number of 3.0×109 pentamer+ T cells could be reached four weeks later, representing a 7800-fold expansion Our data show that allo-restricted T cells with high specificity for tumor-associated antigens can be rapidly generated, sorted and expanded to clinically applicable numbers. [Display omitted]

798. Epstein Barr Virus-Specific Cytotoxic T Lymphocytes (EBV-CTL) Expressing an Anti-CD30 Chimeric T-Cell Receptor (cTcR) for the Treatment of Hodgkin's Disease (HD)

HD is a suitable disease for immunotherapy, and in patients with EBV-related HD, adoptive transfer of EBV-CTLs has produced disease responses. For non-EBV-related HD, the CD30 molecule, which is present on the malignant cells of almost all patients with HD, can be targeted using T-lymphocytes genetically modified to express an anti-CD30 cTcR {Cancer Res,1998;58:1116}. Transgenic T cells expressing a cTcR have been used in clinical trials for HIV patients, but they produced only minimal effects mostly because artificial receptors lack co-stimulation signals required for the full activation of resting T-cells. We have shown that EBV-CTL can fulfill this need, since the co-stimulatory signals delivered by EBV-infected B cells after native receptor engagement ensure full functionality when the CTL subsequently bind to tumor cells through their cTcR {Blood,2002;99:2009}. We first evaluated whether EBV-CTL can be redirected to kill CD30+ HD cell lines and whether they retain their specificity and antigen repertoire. EBV-CTLs were prepared from 8 EBV+ healthy donors using weekly stimulation with irradiated autologous EBV-transformed lymphoblastoid cell lines (LCL) in the presence of IL-2 (50U/mL). CTL were transduced after the 3rd stimulation and were further expanded with 3-4 weekly LCL/IL-2 stimulations. The expansion rate of the transduced CTL was similar to that of control EBV-CTL. Transduced CTL retained killing of their autologous LCL targets through their native receptor (64.4|[plusmn]|16.4% at 20:1 E:T ratio), and became able to lyse CD30+ malignant lymphoma targets through their cTcR (e.g. HDLM=45.4|[plusmn]|16.4% and Karpas-299=42.5|[plusmn]|16.4%). Killing of CD30+ tumor cells was significantly inhibited when target cells where preincubated with an anti-CD30 blocking antibody (16.5|[plusmn]|12%). Of potential concern, however, is that CD30 is also expressed by activated normal T-lymphocytes: expression was undetectable on resting T cells, but increased to 3-32% on day 4-7 after stimulation with autologous LCL. Fortunately, expression dwindles to 3-6% by two weeks as an EBV-specific line emerges, suggesting that CD30 is expressed only in the early phases of T-cell activation. As anticipated from these data, therefore, expression of a CD30 cTcR did not impair the antigenic repertoire of the EBV-CTL, which retained the same pattern of immunodominant MHC class I epitopes (detected by tetramer binding) as control cells. We also performed co-culture experiments to evaluate whether infusion of CTL-CD30 cTcR could cross-compromise the primary reactivation of other virus-specific CTL. Autologous EBV-CTLs engineered to express the CD30-cTCR were added to cultures of PBMC stimulated to reactivate cytomegalovirus- or adenovirus-specific CTL. In 4/4 donors, the percentage of CMV pp65+ T cells did not change, while generation of adenovirus-specific T cells (Hexon-tetramer+) was significantly reduced in only 1/3 donor. These data support the feasibility of using EBV-CTL bearing a cTcR for CD30 to treat EBV negative HD.

Epstein Barr Virus (EBV)-Specific Cytotoxic T Lymphocytes (CTL) Expressing an Anti-CD30 Chimeric T Cell Receptor (CTCR) for the Treatment of Hodgkin's Disease (HD).

HD may be a suitable target for immunotherapy, and in patients with EBV-associated HD, adoptive transfer of EBV-CTL has produced disease responses. An alternative target is the CD30 molecule, which is present on the malignant cells of almost all patients with HD. CD30 is a member of the TNF superfamily and monoclonal antibodies directed to this antigen are currently under investigation in patients with relapsed HD. An alternative way to target CD30 is by the construction of T cells expressing cTcR specific for the antigen. T lymphocytes engineered to express this cTcR can specifically kill CD30+ HD cell lines {Cancer Res , 1998; 58:1116}. However, these chimeric molecules connect the antigen-recognition properties of CD30 antibodies with the endodomain of CD3ζ, which is insufficient to fully activate resting T cells to proliferate and release cytokines. As a consequence chimeric T cells that express these endodomains divide infrequently, lose activity and have performed poorly in-vivo. Full T cell activation requires receptor engagement to be accompanied by a sequence of co-stimulatory stimuli. We have shown that EBV-CTL can fulfill this need, since the co-stimulatory signals delivered by EBV-infected B cells after native receptor engagement ensure full functionality when the CTL subsequently bind to tumor cells through their cTcR. We first evaluated whether EBV-CTL can be redirected to kill CD30+ HD cell lines and whether they retain their specificity and antigen repertoire. EBV-CTLs were prepared from 8 EBV+ healthy donors using weekly stimulation with irradiated autologous EBV-transformed lymphoblastoid cell lines (LCL) in the presence of IL-2 (50U/mL). CTL were transduced after the 3rd stimulation and further expanded with 3–4 weekly LCL/IL-2 stimulations. The expansion rate of the transduced CTL was similar to that of control EBV-CTL. Transduced CTL retained killing of their autologous LCL targets through their native receptor (64.4±16% at 20:1 E:T ratio), and became able to lyse CD30+ malignant lymphoma targets through their cTcR (e.g. HDLM-2=45.4±16% and Karpas-299=42.5±17%). Killing of CD30+ tumor cells was significantly inhibited by preincubation with an anti-CD30 blocking antibody (16.5±12%). Of potential concern, however, is that CD30 is expressed by activated normal T lymphocytes: expression was undetectable on resting T cells, but increased to 3–32% on day 4–7 after stimulation with LCL. Fortunately, expression dwindles to 3–6% by two weeks as an EBV-specific line emerges, suggesting that CD30 is expressed only in the early phases of T cell activation. As anticipated from these data, therefore, expression of a CD30 cTcR did not impair the antigenic repertoire of the EBV-CTL, which retained the same pattern of immunodominant MHC class I epitopes (detected by tetramer) as control cells. We also performed co-culture experiments to evaluate whether infusion of CTL-CD30 cTcR could cross-compromise the primary reactivation of other virus-specific CTL. Autologous EBV-CTLs engineered to express the CD30-cTcR were added to cultures of PBMC stimulated to reactivate cytomegalovirus- or adenovirus-specific CTL. In 4/4 donors, the percentage of CMV pp65+ T cells did not change, while generation of adenovirus-specific T cells (Hexon-tetramer+) was significantly reduced in only 1/3 donor. These data support the feasibility of using EBV-CTL bearing a cTcR for CD30 to treat both EBV+ and EBV− HD.

SAP mediates specific cytotoxic T-cell functions in X-linked lymphoproliferative disease

AbstractCytotoxic T cells (CTLs) and natural killer cells play a major role in the immune response to Epstein-Barr virus (EBV) infection. In X-linked lymphoproliferative (XLP) disease, a severe immunodeficiency, immunodysregulatory phenomena are observed following EBV infection, suggesting that defects exist in these effector populations. The gene defective in XLP is SAP (signaling lymphocytic activation molecule [SLAM]–associated protein), an adaptor protein that mediates signals through SLAM and other immunoglobulin superfamily receptors including 2B4. We generated EBV-specific T-cell lines from controls and XLP patients and examined CTL function in response to different stimuli. We show that XLP patients can generate EBV–T-cell lines that are phenotypically similar to those from controls. XLP patient EBV–T-cell lines showed a significant decrease in interferon-gamma (IFN-γ) production in response to 2B4 and autologous EBV-transformed lymphoblastoid cell line (LCL) stimulation but not in response to SLAM. Furthermore, XLP EBV–T-cell lines demonstrated markedly decreased cytotoxic activity against autologous LCLs. By retroviral gene transfer of the SAP gene into XLP EBV–T-cell lines, we show reconstitution of IFN-γ production and of cytotoxic activity confirming SAP-dependent defects. These studies demonstrate that in XLP the lack of SAP affects specific signaling pathways resulting in severe disruption of CTL function.

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein-Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivites directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416-424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC: peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.

Restoration of tumor-specific HLA class I restricted cytotoxicity in tumor infiltrating lymphocytes of advanced breast cancer patients by in vitro stimulation with tumor antigen-pulsed autologous dendritic cells.

Breast tumor infiltrating lymphocytes (TIL) are enriched in tumor-specific cytotoxic T lymphocytes (CTL), and may represent a superior source of CTL compare to peripheral blood lymphocytes (PBL), for adoptive T cell immunotherapy of breast cancer. However, the immunocompetence of TIL and the possibility to consistently restore their tumor-specific lytic activity in vitro remains an open issue. In this study we evaluated the potential of tumor antigen-pulsed fully mature dendritic cell (DC) stimulation in restoring tumor-specific cytotoxicity in anergic TIL populations from advanced breast cancer patients. In addition we have compared tumor-specific T cell responses induced by tumor antigen-loaded DC stimulation of TIL to responses induced from PBL. Although TIL were consistently non-cytotoxic after isolation or culture in the presence of interleukin-2 (IL-2), in matched experiments from three consecutive patients, tumor-lysate-pulsed DC-stimulated CD8+ T cell derived from TIL were found to be significantly more cytotoxic than PBL (p < 0.05). In addition, cytotoxicity against autologous tumor cells was more significantly inhibited by an anti-HLA class I (W6/32) MAb in TIL compared to PBL (p < 0.05). CTL populations derived from TIL and PBL did not lyse autologous EBV-transformed lymphoblastoid cell lines, and showed negligible cytotoxicity against the NK-sensitive cell line K562. Furthermore, in both CD8+ T cell populations the majority of the tumor-specific CTL exhibited a Th1 cytokine bias (IFN-gamma(high)/IL-4(low)). Taken together, these data show that tumor lysate-pulsed mature DC can consistently restore tumor-specific lytic activity in non-cytotoxic breast cancer TIL. These results may have important implications for the treatment of chemotherapy resistant breast cancer with active or adoptive immunotherapy.

Cytolytic T cell reactivity to Epstein-Barr virus is lost during in vitro T cell expansion.

In the setting of allogeneic hematopoietic stem cell transplantation, ex vivo culturing of donor T lymphocytes is a necessary step for processes such as gene modification. Often the aim is to enable control of undesired alloreactivity after in vivo administration of the cultured cells. However, it is not fully understood how T cell reactivity against donor and third-party targets is affected by the ex vivo cell culturing process. We have assessed how the activity of anti-Epstein Barr virus (EBV)-specific T lymphocytes from healthy EBV-seropositive donors is affected by in vitro cell culturing. Peripheral blood mononuclear cells (PBMCs) were expanded in X-VIVO 15 culture medium supplemented with 5% human serum. The cells were stimulated by either OKT3 (10 ng/ml) and interleukin (IL)-2 (500 U/ml) or by using anti-CD3/CD28-coated immunomagnetic beads and IL-2 (100 U/ml). Induction of polyclonal EBV-specific cytotoxic T lymphocyte cultures was attempted by stimulation of the in vitro-expanded cells at different time points during the cell expansion process, with pre-established autologous EBV-transformed lymphoblastoid cell lines (LCLs). While EBV-specific cytotoxic T lymphocytes (CTL) were generated from untreated PBMCs of 5 healthy donors, EBV-specific cytotoxicity was significantly decreased or absent in CTL cultures established from in vitro-expanded PBMCs. Our results indicate that the ex vivo cell expansion process itself significantly reduces the activity and/or the number of EBV-specific T cells. Additional stimulation with CD28 antibodies could not prevent this effect. Because T cell depleted bone marrow or stem cell grafts are known to contribute to the development of post transplant lymphoproliferative disorders, this should be taken into consideration if one considers expanding and administering PBMCs in conjunction with a T cell-depleted stem cell grafts.

CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.

The major Epstein-Barr virus (EBV) envelope glycoprotein gp340 when incorporated into Iscoms primes cytotoxic T-cell responses directed against EBV lymphoblastoid cell lines

A recombinant form of the EBV envelope glycoprotein and vaccine candidate gp340, lacking its hydrophobic transmembrane region, was incorporated into Iscoms after coupling to phosphatidyl ethanolamine via carbohydrate residues. Coupling by partial oxidation of gp340 carbohydrate with sodium periodate partly denatured the incorporated gp340 as indicated by its reduced reactivity with monoclonal antibodies that recognise the major neutralising epitope. Immunisation of cottontop tamarins with these Iscoms elicited antibody responses to gp340, but these antibodies only poorly recognised the major neutralising epitope in a competition ELISA and were unable to neutralise EBV in vitro. Despite the lack of neutralising antibody, immunisation with these Iscoms primed significant in vitro proliferative responses to soluble gp340 in lymphocytes from the draining lymph nodes and spleen. T-cell lines were raised from both immunised and control animals by in vitro stimulation of peripheral blood lymphocytes or spleen cells with autologous EBV-transformed lymphoblastoid cell lines. The T-cell lines from control animals had higher numbers of CD4+ T-cells than CD8+ T-cells and were not cytotoxic for autologous lymphoblastoid cell lines (LCL). In contrast the lines from immunised animals contained more CD8+ T-cells than CD4+ T-cells and had marked cytotoxicity for autologous LCL.

Assessment and characterization of the cytolytic T lymphocyte response against Epstein-Barr virus in patients with non-Hodgkin's lymphoma after autologous peripheral blood stem cell transplantation.

The cytolytic T lymphocyte (CTL) response has often been used to assess the reconstitution of T cell function after allogeneic or autologous bone marrow transplantation (BMT). Less is known, however, about the reconstitution of the CTL response after peripheral blood stem cell transplantation (PBSCT). Therefore, we investigated the CTL response against Epstein-Barr virus (EBV) of patients undergoing autologous PBSCT. CTLs of six patients with relapsed non-Hodgkin's lymphoma and multiple myeloma were established before and at different times after PBSCT by in vitro stimulation of peripheral blood lymphocytes with autologous EBV-transformed lymphoblastoid cell lines (LCLs). The efficiency of T cell priming by LCLs was assessed at the time of initiation of CTL lines; the proliferative response was strongly reduced during the first 4 months and increased 5 months or more following PBSCT. Cytolytic activity was measured after three or four restimulations of CTLs. All patients investigated had a detectable EBV-specific CTL response which was poor during the first weeks after transplantation, accompanied by a strong non-MHC-restricted cytotoxic activity and a high proportion of CD56-positive T cells. Five or more months after PBSCT, a specific CTL response against EBV was seen which was similar to the situation prior to PBSCT, while the unspecific cytotoxic response decreased. Blocking experiments with monoclonal anti-CD3, anti-CD8 or anti-MHC I antibodies resulted in substantial inhibition of autologous LCL lysis, whereas anti-CD4 or anti-MHC II antibodies had no effect. Finally, autologous PHA blasts of a patient with the HLA haplotype A1/9+, B5/8+, Cw4/7+, were loaded with various EBNA-derived nonapeptides known to be presented by HLA B8 or A11, and exposed to autologous, EBV-directed CTLs. Specific lysis by CTLs only occurred with HLA B8-, but not with HLA A11-restricted nonapeptides. This demonstrated the existence of an MHC I-restricted anti-EBV CTL response after PBSCT. Taken together, the results show that the anlaysis of the EBV-directed CTL activity may serve as a surrogate marker to assess the reconstitution of the cellular immune response in patients undergoing autologous PBSCT.

Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma.

Evasion from cytotoxic T-lymphocyte (CTL) surveillance may be an important step in the pathogenesis of Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) as suggested by the consistent down-regulation of all transformation-associated viral antigens, except EBV nuclear antigen 1 (EBNA-1), and of certain HLA class I alleles in BL biopsies and cell lines that maintain the tumor cell phenotype in vitro. The most common HLA class I defect recorded in BL lines is a selective down-regulation of HLA-A11. To gain some insight into the role of HLA-A11 down-regulation in pathogenesis of BL, we have investigated the target specificity of HLA-A11-restricted CTLs derived by stimulation of lymphocytes from three EBV-seropositive individuals with autologous EBV-transformed lymphoblastoid cell lines. Recombinant vaccinia viruses carrying the coding sequences for EBNA-1, -2A, -2B, -5, -3, -4, and -6 (also known as EBNA-1, -2A, -2B, -LP, -3a, -3b, and -3c, respectively) and EBV latent membrane protein 1 were used to induce high levels of expression of the relevant EBV antigen in fibroblasts derived from HLA class I-matched individuals. EBNA-4-expressing fibroblasts were the predominant target of HLA-A11-restricted CTLs in all three donors. A less pronounced and less regular EBNA-6-specific cytotoxic component was found in two of the donors.

Functional heterogeneity among herpes simplex virus-specific human CD4+ T cells.

One hundred thirteen HSV-specific CD4+ T cell clones were established from the PBL of a healthy person and their functional heterogeneity was investigated. All clones proliferated in response to stimulation with HSV in the presence of autologous APC. Among those, 48 clones showed cytotoxic activity to HSV-infected autologous EBV-transformed lymphoblastoid cell line, but not to HSV-infected autologous fibroblasts, HSV-infected allogeneic cells, or K562 cells (group 1). Five clones showed cytotoxicity against HSV-infected autologous cells as well as HSV-infected allogeneic cells and K562 cells (group 2). The cytotoxicity of these clones was found to be mediated by the direct killing but not by the "innocent bystander" killing of target cells. Sixty clones showed no cytotoxic activity, however, among these, 23 revealed HLA-unrestricted and nonspecific cytotoxicity in the presence of PHA in culture (group 3), and the remaining 37 did not show any cytotoxic activity even in the presence of PHA (group 4). The cytotoxic patterns of these clones did not change in activated and resting phases, suggesting that the difference in cytotoxic ability does not depend on cell cycles. The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR or anti-CD3 mAb to the culture, whereas these mAb had no effect on the cytotoxicity of group 2. All four groups of clones had helper activity for anti-HSV antibody production by autologous B cells. Moreover it was found that all groups of clones simultaneously produced IL-2, IL-4, and IFN-gamma after culture with APC followed by HSV Ag stimulation. The surface phenotype of all clones was uniformly CD2+, CD3+, CD4+, CD8-, CD29+, CD45RA-, but expression of Leu 8 was varied. These data therefore indicate that HSV-specific human CD4+ T cells are classified into at least four groups according to the presence and specificity of cytotoxicity, i.e., Th cells with HSV-specific and HLA-class II-restricted cytotoxicity, Th cells with HLA-unrestricted and nonspecific cytotoxicity, Th cells with lectin-dependent cytotoxicity, and Th cells without cytotoxic activity. The present finding of functional heterogeneity among virus-specific human CD4+ T cells might shed light on the pathogenesis of CD4+ T cell immunodeficiency, such as human retrovirus infections.

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (>95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3 +,4 +,8 −, CD3 +,4 −,8 −, and CD3 −,4 −,8 − clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.

Down-regulation of class I HLA antigens and of the Epstein-Barr virus-encoded latent membrane protein in Burkitt lymphoma lines.

Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) cells are relatively or completely resistant to the lytic effect of major histocompatibility complex class I HLA antigen-restricted cytotoxic T lymphocytes (CTLs) generated by stimulating lymphocytes of EBV-seropositive donors with the autologous EBV-transformed lymphoblastoid cell line (LCL). We previously found that EBV-negative and EBV-carrying BL lines derived from HLA-A11-positive donors were not only resistant to lysis by the HLA-A11-restricted CTL generated by stimulation with the autologous LCL, but also to HLA-A11-specific CTL derived from lymphocytes of an EBV-seronegative donor stimulated with an allogeneic LCL. Using the same and additional cell lines, we now show that the CTL resistance of the BL lines is probably due to a selective down-regulation of HLA-A11. We also show that the EBV-encoded latent membrane protein is expressed at a lower level in the EBV-carrying BL lines than in EBV-transformed LCLs. Only one of eight in vitro EBV-converted BL lines that shifted to a more LCL-like growth pattern expressed LMP at a high level. This line also reexpressed the HLA-A11 antigen that was undetectable in its EBV-negative progenitor. Our findings suggest that the typical BL cell phenotype is associated with low expression of both proteins.

Multiple HLA class I‐dependent cytotoxicities constitute the “non‐HLA‐restricted” response in infectious mononucleosis

Primary Epstein-Barr virus (EBV) infection, when manifest as infectious mononucleosis (IM), induces a broad-ranging and apparently non-HLA-restricted cytotoxic response whose nature has not been resolved. In the present experiments the ability to cryo-preserve IM mononuclear cell preparations, after depletion of CD16+ natural killer cells, has allowed detailed analysis of the response on appropriately constructed target cell panels. The results show that IM effector preparations are polyclonal with separate HLA class I antigen-dependent reactivities against the autologous EBV-transformed lymphoblastoid cell line (LCL) and particular HLA class I-mismatched LCLs. The autologous LCL-directed response shows the hallmarks of immunologically specific T cell cytotoxicity; only EBV+ B cell blasts are recognized and the interaction can be blocked by monoclonal antibodies to CD3 and CD8 on the effector cell surface and to HLA class I antigens on the target cell. Such findings demonstrate, for the first time, that the primary cytotoxic response to EBV infection includes a virus-specific HLA-restricted component like that found in the T cell memory of persistently infected individuals. Separate components of the response are preferentially active against some (but not all) HLA-mismatched LCLs, the pattern of reactivity being distinct for each individual IM patient and reproducible on repeated testing. Monoclonal antibody blocking experiments show that these HLA-mismatched interactions also involve CD3 and CD8 antigens on the effector cell and HLA class I antigens on the target cell. Where tested, such lysis affected both EBV+ and EBV- B cell blasts from the relevant HLA-mismatched donors. We postulate that a widespread primary infection of the B cell system by EBV leads to a generalized expansion not just of the virus-specific response but also of other T cell responses coincidentally active at the time. The unusual activity of IM effector preparations against HLA-mismatched LCLs arises from fortuitous cross-recognition of allogeneic cells by immunologically specific cytotoxic T cell clones coincidentally expanded in vivo alongside the EBV-specific response.

Expression of target antigen for Epstein-Barr virus-specific cytotoxic T cells on BJAB cells freshly infected with EBV.

The target antigen for Epstein-Barr virus (EBV)-specific cytotoxic T cells (Tc) was expressed on BJAB cells exposed to the B95-8 strain of EBV for at least one hour. Ultraviolet-light (UV)-irradiated noninfectious B95-8 virus also induced the target antigen on BJAB cells. Cold target competition tests suggested that the target antigen expressed on EBV-infected BJAB cells was distinct from the lymphocyte-detected membrane antigen (LYDMA) which was also recognized by the EBV-specific Tc and expressed on autologous EBV-Transformed lymphoblastoid cell line (LCL) cells. Neither of these target antigens for EBV- specific Tc was detected on the surface of EBV-genome positive BJAB cells which had been kept in a long term culture after EBV-infection. Thus, the virion antigen, especially the EBV-membrane antigen (MA), is a possible candidate for the target antigen expressed on EBV-infected BJAB cells. Lysis of EBV-infected BJAB cells was inhibited by target cell treatment with anti-beta 2 microglobulin (anti-beta 2M) antibody and induction of the effector Tc was dependent on the donor individual. These results suggested the possibility that the Tc recognizing EBV-infected BJAB cells are restricted by the major histocompatibility complex (MHC).