EBV-specific CTL Research Articles

Demographics and treatment outcomes in patients with EBV+ PTLD treated with off-the-shelf EBV-specific CTL under an ongoing expanded access program in Europe: First analyses.

7530 Background: Patients undergoing allogeneic hematopoietic cell transplant (HCT) or solid organ transplant (SOT) are at risk of developing Epstein–Barr virus driven post-transplant lymphoproliferative disorder (EBV+ PTLD), a rare hematologic malignancy, which is often aggressive and life-threatening. Patients with relapsed or refractory (r/r) EBV+ PTLD have few treatment options with poor outcomes, demonstrating a clear unmet medical need. Tabelecleucel is an investigational, off-the-shelf, allogeneic EBV-specific T-cell immunotherapy being studied in patients with serious EBV+ diseases (NCT04554914 & NCT03394365) that has demonstrated clinical benefit and favorable safety profile in the treatment of EBV+ PTLD after failure of rituximab (R) ± chemotherapy (Prockop, EBMT 2021, ATC 2021, ASH 2021). Methods: Atara Biotherapeutics supports an ongoing expanded access program (EAP) in Europe for patients with EBV+ diseases who have no other treatment options. Here we report demographics, efficacy and safety results of r/r EBV+ PTLD patients following SOT or HCT who presented between Jul 2020 and Nov 2021 and consented to research. Results: A total of 48 EAP requests from 9 countries for patients with EBV+ diseases were received. Twenty-two patients from 7 countries consented to this research: 16 EBV+ PTLD and 6 EBV+ non-PTLD. Of the 16 PTLD patients 15 received at least one dose of tabelecleucel. Overall, 9 out of 15 (60%) patients achieved a response as assessed by the treating physician, with 6 complete responses and 3 partial responses. Eight out of nine responses were seen after the first cycle. No adverse events were reported as related to tabelecleucel by the treating physician. Conclusions: The successful execution of this European EAP demonstrates the feasibility to deliver an off-the-shelf allogeneic EBV+ T-cell therapy in time-sensitive clinical situations when no other treatment options exist. These data show clinically meaningful outcomes for patients with r/r EBV+PTLD post-SOT or post-HCT treated with tabelecleucel consistent with previously reported favorable safety and efficacy profile (Prockop, ASH 2021).[Table: see text]

Vaccination - a Novel Strategy to Improve the Persistence of CD19CAR Transduced T-Cells in Relapsed Paediatric ALL: Preliminary Results from the CD19TPALL Study

IntroductionPatients with acute lymphoblastic leukaemia (ALL) relapsing after allogeneic stem cell transplantation (SCT) have a dismal prognosis. Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) incorporating co-stimulatory domains for improved persistence and expansion report unprecedented anti-leukemic responses. However, responses are associated with Cytokine Release Syndrome (CRS) due to supra-physiological activation of the redirected T-cells. As an alternative, we studied use of donor-derived Epstein Barr virus (EBV)-specific T cells (CTL) transduced with a 1st generation CD19CAR as effectors, relying on signalling through the endogenous T cell receptor (TCR) to drive more physiological proliferation and persistence. This has enabled us to investigate a novel strategy to facilitate the expansion/persistence of CD19CAR T cells by vaccination with irradiated donor-derived, EBV transformed lymphoblastoid cell lines (LCL). We are conducting a European multi-centre phase I/II study of this approach in patients with pediatric ALL relapsing after SCT and report our interim findings.MethodsDonor-derived EBV-specific CTL were generated from 80mls donor blood by repetitive stimulation with LCL, followed by transduction with an SFG retroviral vector encoding a CD19CAR consisting of the FMC63 single chain Fv linked to a CD3ζ endodomain. Patients were eligible for CD19CAR CTL therapy either pre-emptively if they became MRD-positive (> 5 x 10-4 in BM) within the 1st year post-SCT or prophylactically at day 60-70 post-2nd SCT. All patients had early withdrawal of immunosuppression and received lymphodepletion with fludarabine 90 mg/m2. Patients with detectable residual disease also received cytoreduction with vincristine/dexamethasone prior to infusion of cryopreserved CD19CAR CTL. Persistence of CAR CTL was measured by quantitative PCR and flow cytometry of blood. Disease status was assessed by morphology and IgH MRD analysis on bone marrow samples. The study design incorporated an interim analysis, allowing for vaccination with irradiated LCL if CD19CAR CTL were not detectable in 50% of patients at 2 months post-infusion.ResultsSo far, 20 patients have been recruited (14 pre-emptive, 6 prophylactic arm) and 7 patients treated (3 pre-emptive, 4 prophylactic). The infused cell dose was 2 x 108/m2 in 6 patients and 4 x 107/m2 in the other. CD19CAR expression varied from 12.1-58.9%.No grade 3-5 toxicity was noted. In particular, no CRS, neurotoxicity or graft versus host disease (GVHD) attributable to CD19CAR CTL was seen. B-cell depletion was transient, lasting 1-2 months.In terms of disease response, 2 patients treated prophylactically remain in MRD negative remission after 3 and 17 months’ follow-up. A further patient showed transient clearance of BM MRD following immunotherapy in association with EBV viremia. He subsequently relapsed but has stable disease after retreatment with CD19CAR CTL with LCL vaccination. The other 4 patients had disease progression between 2 weeks and 3 months post-CD19CAR CTL infusion. At a median follow-up of 8 months, 2 patients have died of relapse, 3 are alive with disease and 2 remain disease-free.A planned interim analysis of the initial 6 patients treated with CD19CAR CTL alone showed poor expansion/persistence of CD19CAR CTL which were only detectable in the blood in 1 patient up to 28 days post-infusion. This may reflect that only 1 patient had EBV viremia at the time CD19CAR CTL were infused. In view of this, a second trial cohort received subcutaneous vaccination with irradiated, donor-derived LCL at 2 days before and at 1 and 2 months following CD19CAR CTL infusion to provide signalling through the endogenous EBV-specific TCR. So far, 2 patients have been vaccinated and a 3rd is planned shortly. Data on the effect of vaccination on CD19CAR CTL expansion/persistence will be presented.ConclusionsThis ongoing study shows safety of adoptive immunotherapy with donor EBV CTL transduced with a 1st generation CD19CAR in paediatric patients with ALL relapsing post allo-SCT. However, in the absence of a co-stimulatory domain in vivo expansion and persistence of transferred CTL is poor. We are investigating whether vaccination with irradiated, donor-derived EBV LCL improves persistence and efficacy of CAR transduced T cells and initial data on this approach will be presented DisclosuresPule:Cellectis: Martin Pule’s laboratory receives funding for contract research from Cellectis Therapeutics Other.

Successful treatment of a classic Hodgkin lymphoma‐type post‐transplant lymphoproliferative disorder with tailored chemotherapy and Epstein–Barr virus‐specific cytotoxic T lymphocytes in a pediatric heart transplant recipient

CHL type is the least common major form of EBV-related PTLD but rarely occurs in pediatric recipients; development of CHL subsequent to other PTLD subtypes in the same transplant recipient is even more unusual. Because of its rarity, indications on the best treatment strategy are limited. Patients have been mostly treated with standard HL chemotherapy/radiotherapy, and prognosis seems more favorable than other monomorphic PTLDs. Herein, we describe a pediatric case of EBV-associated, stage IV-B, CHL arising in a heart allograft recipient eightyr after diagnosis of B-cell polymorphic PTLD. The patient was successfully treated with adjusted-dose HL chemotherapy and autologous EBV-specific CTL, without discontinuation of maintenance immunosuppression. At twoyr from therapy completion, the patient is in CR with stable organ function. With this strategy, it may be possible to reproduce the good prognostic data reported for CHL-type PTLD, with decreased risk of organ toxicity or rejection.

Epstein–Barr Virus Infection and Posttransplant Lymphoproliferative Disorder

Epstein-Barr virus (EBV) is an important pathogen in recipients of solid organ transplants (SOT). Infection with EBV manifests as a spectrum of diseases/malignancies ranging from asymptomatic viremia through infectious mononucleosis to posttransplant lymphoproliferative disorder (PTLD). EBV disease and its associated PTLD is more frequently seen when primary EBV infection occurs after transplant, a common scenario in pediatric SOT recipients. Intensity of immunosuppressive therapies also influences the risk for PTLD. The use of EBV viral load monitoring facilitates the diagnosis and management of EBV/PTLD as well as being used to inform preemptive therapy with reduction of immunosuppression, the most effective intervention for prevention of and treatment for PTLD. Other therapies, including the rituximab (anti-CD20 monoclonal antibody) and traditional chemotherapy, are also useful in the treatment of established PTLD. The future development of standards for management based on EBV viral load and routine monitoring of EBV-specific CTL responses promise further improvement in outcomes with EBV and PTLD.

1085 Generation of EBV-specific CTL Lines Enriched in BARF1 Specificities for Improved Adoptive Immunotherapy of Nasopharyngeal Carcinoma

1085 Generation of EBV-specific CTL Lines Enriched in BARF1 Specificities for Improved Adoptive Immunotherapy of Nasopharyngeal Carcinoma

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation

AbstractWe evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

Immunotherapy for EBV-associated malignancies

Since 1995 to date, more than 250 patients with EBV-related diseases received virus-specific CTL. Cell therapy proved to be safe and effective, and achieved some complete remissions also in patients who failed all previous standard treatments. The first clinical results with EBV-specific CTL were obtained for both prophylaxis and treatment of post-transplant lymphoproliferative disease arising in stem cell transplant or solid organ transplant recipients. Based on such encouraging results, the same approach was then extended to other EBV-related diseases, namely Hodgkin's lymphoma, nasopharyngeal carcinoma, and chronic active infection. Nowadays, the modification of the CTL generation protocols and the introduction of new specificities into EBV-specific CTL lines by chimeric antigen receptor transfer allow targeting other viral infections and also non-EBV related malignancies. Aim of this review is to summarize clinical results obtained thus far in adoptive cell therapy approaches with EBV-specific CTL. Moreover, by analyzing ongoing clinical trials, we also provide some insights on the potential future of a successful and paradigmatic history.

Impact of γ-chain cytokines on EBV-specific T cell cultures

BackgroundRecent preclinical adoptive immunotherapy studies in murine models prompt to employ "proper" rather than "as many as possible" antigen-specific T cells to gain better therapeutic results. Ideally, "proper" T cells are poorly differentiated in vitro, but retain the capacity to fully differentiate into effector cells in vivo, where they can undergo long-term survival and strong proliferation. Such requirements can be achieved by modifying culture conditions, namely using less "differentiating" cytokines than IL-2.MethodsTo evaluate this issue in human T cell cultures, we exploited a well characterized and clinical-grade protocol finalized at generating EBV-specific CTL for adoptive immunotherapy. In particular, we studied the impact of IL-7, IL-15 and IL-21 compared to IL-2 on different aspects of T cell functionality, namely growth kinetics, differentiation/activation marker expression, cytokine production, and short-term and long-term cytotoxicity.ResultsResults disclosed that the culture modifications we introduced in the standard protocol did not improve activity nor induce substantial changes in differentiation marker expression of EBV-specific CTL.ConclusionsOur data indicated that the addition of γ-chain cytokines other than IL-2 for the generation of EBV-specific T cell cultures did not produce the improvements expected on the basis of recent published literature. This fact was likely due to the intrinsic differences between murine and human models and highlights the need to design ad hoc protocols rather than simply modify the cytokines added in culture.

Cytolytic T-cell response against Epstein-Barr virus in lung cancer patients and healthy subjects

BackgroundThis study aimed to examine whether EBV seropositive patients with lung cancer have an altered virus-specific CTL response, as compared to age-matched healthy controls and whether any variation in this response could be attributed to senescence.MethodsPeripheral blood mononuclear cells from lung cancer patients, age-matched and younger healthy individuals were used to measure EBV-specific CTLs after in vitro amplification with the GLCTLVAML and RYSIFFDYM peptides followed by HLA-multimer staining.ResultsLung cancer patients and aged-matched controls had significantly lesser EBV-specific CTL than younger healthy individuals. Multimer positive populations from either group did not differ with respect to the percentage of multimer positive CTLs and the intensity of multimer binding.ConclusionsThis study provides evidence that patients with lung cancer exhibit an EBV-specific CTL response equivalent to that of age-matched healthy counterparts. These data warrant the examination of whether young individuals have a more robust anti-tumor response, as is the case with the anti-EBV response.

Four Cases of Chronic Active Epstein-Barr Virus Infection That Performed Reduced-Intensity Conditioning Bone Marrow Transplantation.

Abstract 4734Epstein-Barr virus (EBV) is B cell tropic virus. However, the EBV-infected T and NK cells play a critical role on the pathogenesis of chronic active EBV infection (CAEBV). The diagnostic criteria are fever, lymphadenopathy and hepatosplenomegaly with abnormal antibody titer to EBV and increased amount of the EBV genome in the blood. But the mechanism on how EBV infects T and NK cells is not fully known yet. Once leukemic transformation occur the prognosis of the disease becomes very poor. The evidence that hematopoietic stem cell transplantation (SCT) is effective on CAEBV has accumulated, however, controversy still remains on the necessity of pre-transplant chemotherapy, and what kind of conditioning regimen is suitable during SCT.In this article, we reported 2 cases of T cell type CAEBV and 2 cases of NK cell type CAEBV that have been treated by bone marrow transplantation (BMT) with reduced intensity regimen (RIC) without pre-transplant chemotherapy. RIC consists of Cyclophosphamide (CY, 100-120 mg/Kg), Fludarabine (Flu, 120-150 mg/m2) and low dose TBI (3-4 Gy).Case 1 is an 8-year-old boy with EBV infected CD4 positive T cells. He showed elevated serum enzyme, coronary artery aneurysm, generalized rash, and positive EBV DNA in cerebrospinal fluid. The EBV genome in peripheral mononuclear cells was 1.3+105 copies /μgDNA. Southern blot analysis with EBV terminal repeat probe revealed clonal proliferation of virus. Tetramer analysis did not detect EBV specific CTL. He received modified HLH 2004 protocol (Henter et al, 2006). Immediately after normalization of liver dysfunction, BMT were performed from unrelated donor. Now he is in complete remission.Case 2 is an 8-year-old boy with EBV infected CD4 positive T cells. He also showed liver dysfunction, lymphadenopathy and positive EBV DNA in cerebrospinal fluid. The EBV genome in peripheral mononuclear cells is 2.6+105 copies /μgDNA. EBV clonality was detected by Southern bolt analysis. Tetramer analysis of BRLF1, EBNA3A and EBNA3B peptides showed EBV specific CTL. BMT from HLA-matched sibling donor achieved complete chimeras. But the EBV genomes in his peripheral blood increased to 4.7 +103 copies /μgDNA after BMT. EBV positive CD4 T cells were present. At the moment, it is not clear whether the origin of EBV positive T cells is from donor or not.Case 3 is a 21-year-old female with mosquito allergy with EBV infected NK cells. Liver dysfunction and lymphadenopathy were occasionally complained. The EBV genome in her peripheral blood is 1.2+105 copies /μgDNA. EBV showed monoclonal amplification. BRLF1 specific CTL were detected by tetramer analysis. HLA one antigen mismatched BMT from unrelated donor induced complete remission after grade 3 skin GVHD.Case 4 is a 19-year-old boy with CAEBV secondary to EBV-hemophagocytic lymphohistiocytosis. EBV positive NK cells were present. EBV genome in his peripheral blood was 1.2+104 copies /μgDNA. EBV showed monoclonal amplification. EBV specific CTL were not detected. After BMT from HLA-matched unrelated donor, EBV DNA was rapidly eliminated from his blood. But he suffered from pure red cell aplasia caused by major Blood type incompatibility and now treated with Rituximab.Four cases of CAEBV were described. RIC was demonstrated to be effective for engraftment of donor bone marrow cells without any serious adverse effects except pure red cell aplasia. Addition of pre-transplant chemotherapy seemed to be non-essential for engraftment or remission induction. In case 2 EBV infected peripheral CD4 positive cells were demonstrated after BMT. Determining the origin of the EBV infected CD4 positive T cells may provide hints for understanding on how EBV infects T cells or how EBV infected Tcells survive after BMT. Disclosures:No relevant conflicts of interest to declare.

Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients

Treatment of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of autologous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at (http://www.clinialtrials.gov) as NCT00608257.

Immunological Function against Human T-Lymphotrophic Virus Type I in Carriers with Collagen Diseases and HAM/TSP.

Human T-cell leukemia virus-1 (HTLV-1) is the first human retrovirus that has infected approximately 10–20 million people worldwide and causes adult T cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) after long-term chronic infection. The immune response to HTLV-1 is typically enhanced in HAM/TSP, while impaired cell-mediated immunity has been identified as a causative basis of immunodeficiency in ATLL. The negative immuno-regulatory program death-1 (PD-1) signaling is involved in auto-immunity, allergy, sites of immune privilege, and antitumor immunity. We have previously reported decreased frequency and function of HTLV-1 Tax-specific CD8+ T-cell in ATLL patients due to insufficient cytolytic effector molecules, as well as upregulated PD-1 expression on HTLV-1-specific CTLs from asymptomatic carriers (ACs) and ATLL patients. That PD-1 expression was significantly higher on HTLV-1 specific CD8+ T-cells, as well as CMV− and EBV-specific CTLs in ATLL patients compared to ACs, suggested that PD-1 signaling plays a role in fostering persistent HTLV-1 infections, facilitating immune evasion by ATLL cells, which may further ATLL development (ASH annual meeting 2006, 2007). However, there is no report on the repertoir and PD-1 expression of HTLV-1-specific CTL in carriers with hyper immune states such as collagen disease or HAM/TSP. Therefore, to characterize HTLV-1-specific CTL in asymptomatic HTLV-1 carriers (ACs), carriers with collagen disease (CCs) and HAM/TSP patients, we examined the frequency, diversity and PD-1 expression of HTLV-1-specific CTL in 23 ACs, 33 carrier with collagen vascular disease, 40 HAM/TSP patients and 18 healthy donors (HDs) using 16 distinct HTLV-1 Tax/Env HLA-A*0201 and HLA-A*2402 tetramers. The difference in frequency of HTLV-1 Tax-specific CTL positivity was not statistically significant between CCs, HAM/TSP or ACs. In contrast, HTLV-1 Env-specific CTL were significantly more frequent in autoimmune disorders and HAM/TSP than those in ACs (Table 1). On the other hand, percentages of CD8+ lymphocytes from these carriers expressing PD-1 were significantly higher than that from HDs. Futhermore, the rates of PD-1 expression on CMV- or EBV-specific CTL in CCs, HAM/TSP and ACs were similar although PD-1 expression was significantly down-regulated on HTLV-1-specific CTL in CCs and HAM/TSP compaired to ACs (Figure1, p<0.05 and p<0.01, respectively). Few lymphocytes from these carriers were positive for the PD-L1 ligand, while PD-L1 expression was observed on lymphocytes cultured for 12 hours from ACs (5 of 6), but less from CCs and HAM/TSP (1 of 6 and 0 of 4, respectively). The diversity, frequency and repertoir of HTLV-1-specific CD8+ T cell clones, especially HTLV-1 Env CTLs, may be related to the hyper immune response in CCs and HAM/TSP. The down-regulation of PD-1 on HTLV-1 Tax-specific CTL and loss of PD-L1 expression in CCs and HAM/TSP may explain the apparent hyper immune response against HTLV-1 Tax protein.Table 1. HTLV-1/HLA tetramers positive subjectsHLA alleleTetramersACCCHAM/TSPA*02Tax100% (8/8)75% (9/12)90% (9/10)A*02Env0% (0/8)25% (3/12)10% (1/10)A*24Tax94% (17/18)88% (21/24)100% (15/15)A*24Env6% (1/18)63% (15/24)*40% (6/15)*Tax CTL96% (22/23)82% (27/33)100% (18/18)Eav CTL4% (1/23)48% (16/33)*50% (9/18)*The number of subjects positive for tetramers; the percentages of HTLV-1/HLA tetramer+ CD8+ T cells in the CD8+ lymphocytes > 0.1% are counted as subjects positives for tetramer, *, P < 0.01, significant differences from AC by χ2 test. [Display omitted]

Complete Tumor Responses in Lymphoma Patients Who Receive Autologous Cytotoxic T Lymphocytes Targeting EBV Latent Membrane Proteins

EBV-associated Hodgkin's Lymphoma (HL) and some non-Hodgkins lymphoma (NHL) have type II viral latency expressing the subdominant EBV antigens EBNA1, LMP1 and LMP2. These antigens may serve as targets for immunotherapy approaches and in previous studies, we used polyclonal EBV-specific CTL in patients with relapsed EBV +ve HL obtaining 2 complete and 1 partial response in 11 patients. Analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL enriched for effector cells specifically targeting LMP antigens would have greater efficacy in these patients. LMP-CTL were generated using dendritic cells for initial stimulations then EBV-transformed lymphoblastoid cell lines (LCL) both of which had been genetically modified to overexpress either LMP2 alone or inactive LMP1 (ΔLMP1) and LMP2 by transduction with an Ad5f35LMP2 (n=16) or Ad5f35ΔLMP1-I-LMP2 (n=14) vector respectively. All LMP-CTL lines were polyclonal comprising CD4+ (mean 17±18%; range 1–92%) and CD8+ (mean 74 ± 25%; range 1–99%) T-cells. Flow cytometric analysis of memory markers revealed mixed populations of CD45RA- CD62L- T-cells (45±15%; range 31–63%) and CD45RA- CD62L+ T-cells (34±5%; range 28–41%). The CTL lines had specificity for CD4+ and CD8+ restricted LMP2 epitopes alone (n=19; mean 1; range 0–7) or both LMP1 and LMP2 epitopes (n=13; mean 2; range 0–6) per CTL line, as determined using overlapping LMP1 and LMP2 peptide pools in ELISPOT assays. Twenty-four patients with EBV+ Hodgkin's Lymphoma and non-Hodgkin Lymphoma have been treated on dose escalation studies. 16 with LMP2 CTLs and 8 with LMP1/2 CTLs. No immediate toxicity was observed. After CTL infusion, increased numbers of LMP-specific T cells were detected in the blood of 15/22 evaluable patients, (range 2 to 70 fold) persisting for up to 3 months. Additionally, two patients had lymph node biopsies 3–6 months post CTL, which showed selective accumulation of LMP2-multimer positive cells in lymph nodes. 12/13 high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment after chemotherapy remain in remission for a median of 2years (range >3months to >5years) after CTL. 11 patients had detectable disease at the time of CTL, 2 of these had progressive disease by 8 weeks and 9 had clinical responses. The median duration of the clinical responses is 1 year with one stable disease (>12months), one partial response (36 months) and 7 complete responses (range 9 months to >4.5 years). One of the complete responders was biopsied 7 weeks after receiving CTL, which were predominantly CD4+ (92%). Increased CD4+ T cells were seen compared to pre-CTL biopsy specimens and imaging studies confirmed remission. In conclusion, immunotherapy with CTL targeting LMP antigens is well tolerated in patients with EBV+ lymphoma and infused LMP-CTL can accumulate at tumor sites and induce complete and sustained clinical responses.

CTL Exhaustion in Persistent HTLV-1 Infection and ATLL Is Restored through PD-1/PD-L1 Pathway.

Adult T-cell leukemia/lymphoma (ATLL) develops after infection with human T-cell leukemia virus-1 (HTLV-1) with a long latent period. The negative regulatory programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway has been implicated in the induction of CTL exhaustion during chronic viral infection and tumor escape from host immunity. We have previously reported decreased frequency and function of HTLV-1 Tax-specific CD8+ T-cell in ATLL patients due to insufficient cytolytic effector molecule. To determine whether the PD-1/PD-L1 pathway is involved in the immunosuppressive state in HTLV-1-infected individuals and immune evasion in ATLL patients, we examined PD-1/PD-L1 expression in asymptomatic HTLV-1 carriers (ACs) and ATLL patients. PD-1 expression was significantly up-regulated on lymphocytes in ACs and ATLL patients compared to non-HTLV-1-infected individuals. The rates of PD-1 expression on HTLV-1-specific CTL in ACs and ATLL patients were similarly high (p = 0.32) although PD-1 expression was significantly up-regulated on total lymphocytes, cytomegalovirus- and EBV-specific CTL in ATLL patients compared to ACs and non-HTLV-1-infected individuals (Figure1, p < 0.01). This may be one reason why HTLV-1-infected cells are not eradicated even in the presence of anti-HTLV-1 antigen-specific CTL in the carrier state. Primary ATLL cells in 15.0% of ATLL patients and an HTLV-1-infected cell line expressed PD-L1, while few lymphocytes from ACs were positive for this ligand. Anti-PD-L1 antagonistic antibody restored HTLV-1-specific CTL functions (IFN-γ, TNF-α, CD107a) ex vivo (Figure2). These observations suggest that the immunoregulatory PD-1/PD-L1 pathway is operative during persistent viral infection, and the pathway may induce the exhausted phenotype of CTL in HTLV-1 infected individuals, which may lead to immunodeficiency and immune evasion in ACs and ATLL patients. Furthermore, reactivation of exhausted T cells by blocking the PD-1/PD-L1 may be an effective immunological strategy for the clearance of persistent HTLV-1 infection and the treatment of ATLL. [Display omitted] [Display omitted]

Gene Transfer of IL-7R alpha (IL-7Rα) on Antigen-Specific Cytotoxic T Cells (CTLs) Restores Their Ability To Respond to IL-7 Cytokine.

Systemic administration of IL-2 may improve the expansion and persistence of adoptively transferred anti-tumor CTLs. However, significant toxicity and concomitant expansion of regulatory T cells (Tregs) limit the clinical value of this strategy. IL-7 plays a crucial role in maintaining T-cell homeostasis, and administration appears well tolerated and not to expand Tregs. Nonetheless, the value of IL-7 for promoting proliferation of tumor-specific CTLs after adoptive transfer remains questionable, due to the lack of expression of the IL-7 receptor (IL-7Rα) on effector CTLs. Thus we have found IL-7Rα to be essentially undetectable in our model of EBV-specific CTL lines (EBV-CTLs) due to downregulation of IL-7Rα transcripts. To determine whether transgenic expression of IL-7Rα can restore the capacity of established EBV-CTLs to respond to IL-7, the full-length of IL-7Rα was cloned in the SFG retroviral vector (SFG/IL-7Rα). We transduced EBV-CTLs generated from 5 healthy donors and evaluated their growth kinetics and antigen specificity. As control, EBV-CTLs were transduced with the SFG retroviral vector encoding for a truncated form of the CD34 molecule (SFG.ΔCD34). After transduction with SFG/IL-7Rα, IL-7Rα was detectable in 58% to 76% of EBV-CTLs. The transgenic IL-7Rα was functional since addition of IL-7 (2ng/mL) to transgenic cells induced phosphorylation of STAT5 within 10 min. To evaluate whether rhIL-7 sustained the expansion of EBV-CTLs/IL-7Rα+, transduced CTLs were stimulated weekly with autologous EBV-LCLs in the presence of IL-2 (50U/mL) or IL-7 (2ng/mL). Control EBV-CTLs and EBV-CTLs/IL-7Rα+ expanded equally well with IL-2. However, only EBV-CTL/IL-7Rα+ significantly proliferated in the presence of IL-7 [from 1×106 cells to 103×106 cells (range, 38-286×106)] over a period of five weeks. CTL expansion remained antigen dependent, and antigen withdrawal led to cessation of CTL growth. EBV-CTLs/IL-7Rα+ showed the expected growth advantage in the presence of IL-7, increasing from 55%±15% to 79%±5% of the total within 5 weeks. In contrast, the proportion of IL-7Rα+ cells marginally declined when the cells were expanded with IL-2 (from 64%±14% to 40%±11%). Importantly, EBV-CTLs/IL7Rα+ expanded with IL-7 retained their ability to respond to other common-γ-chain cytokines. EBV-CTLs/IL7Rα+ grown with IL-7 for more than 2 weeks, expanded further when exposed to both antigen and IL-2 or IL-15. Control CTLs grown with IL-2 and EBV-CTLs/IL-7Rα+ grown with IL-7 both remained polyclonal, as assessed by VβTCR repertoire analysis, and were mostly CD3+/CD8+ (> 90±8%) with an effector-memory profile. EBV-CTLs/IL-7Rα+ also retained antigen specificity measured by tetramer staining, by IFNγ release in response to EBV-peptides and by MHC-restricted killing of autologous LCLs (52%±18% at a ratio 20:1 vs 9±17% of allogeneic LCL); this killing was comparable to the activity of control CTLs cultured in IL-2 (51%±27%). Preliminary studies indicate that EBV-CTLs/IL-7Rα+ also expand in vivo in response to IL-7 and maintain their anti-tumor activity in a SCID mouse xenograft model.

Immunity, Homing and Efficacy of Allogeneic Adoptive Immunotherapy for Posttransplant Lymphoproliferative Disorders

Adoptive immunotherapy using autologous Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes (auto-CTL) can regress posttransplant lymphoproliferative disorders (PTLD). Widespread applicability of auto-CTL remains constrained. Generation is time-consuming, and auto-CTL cannot be established in patients treated with the B-cell depleting antibody rituximab. By contrast, pregenerated allogeneic CTL (allo-CTL) offers immediate accessibility. Allo-CTL has previously shown efficacy in "early" polyclonal- PTLD. We treated three patients with aggressive, advanced monoclonal-PTLD following solid-organ transplantation. All were refractory to at least three prior therapies. Despite HLA disparity, there was negligible toxicity, with early in vivo antiviral efficacy and reconstitution of EBV peptide-specific immunity. Two patients attained complete remission (CR). One remains in CR 17 months following therapy, coincident with persistence of donor-derived tumor targeted EBV-specific CTL; the other died of non-PTLD related pathology. In the third patient, autopsy demonstrated homing of allo-CTL at the tumor site. Larger prospective studies of EBV-specific allo-CTL in PTLD are warranted.

Human antigen presenting cells stimulate HBV specific CTL and Th cells in the trimera mouse model

Introduction: The strength and breadth of antiviral T cell response is correlated with disease outcome from hepatitis B. Thus, induction of HBV-specific T cells by therapeutic vaccination might represent a new therapeutic strategy, as demonstrated in the humanized trimera mouse model. However, nod.scid mouse derived antigen presenting cells (APC) might reconstitute together with transferred human B cells, monocytes and dendritic cells. Thus, it was hypothesized that murine APC might crosspresent vaccine derived epitopes to human T cells. Methods: To study this issue, Balb/c mice were lethally irradiated, reconstituted with nod.scid mouse bone marrow and transplanted with human PBMC from volunteers or chronic HBV carriers. Human PBMC were transferred either unmanipulated or depleted for total HLA DR+ APC, CD14+ monocytes, CD19+ B cells or BDCA–1+/–4+ DC. Vaccination of such trimera was performed i.p. with the Th cell antigens tetanus toxoid and recombinant HBV core (HBc) antigen, or with the synthetic CTL epitopes HBc18–27 and EBV280–288. Mouse APC were assessed by FACS analysis. Antigen specific human Th cell and CTL frequencies were analysed by IFNg ELISpot. Human cytokines were studied by cytokine bead array. Results: Very low but increasing numbers of mouse B cells and monocytes were detected in peritoenum, spleen and bone marrow of trimera mice. The only human cytokines detectable in peritoneal lavage were IL–1b, IL–6, IL–8 and IFNg. Very strong HBc and EBV specific Th cell and CTL responses were induced in vaccinated trimera mice implanted with total PBMC. Depletion of total DR+ APC, or BDCA–1+/–4+ DC led to abrogation of Th cell and CTL responses, demonstrating the need of human DC for T cell stimulation in trimera mice. In contrast, monocyte and B cell depletion only partly reduced HBc and EBV specific T cell responses. Conclusions: All three human APC populations contribute to effective stimulation of HBV specific human T cell responses in trimera mice, although only DC are indispensable. Mouse derived APC seem not to play a functionally relevant role in this mouse model. Thus, effective vaccination approaches in trimera mice should be predictive of similar responses in patients.

T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNγ production

EBV is associated with a broad range of malignancies. Adoptive immunotherapy of these tumors with EBV-specific CTL proved useful. We generated a panel of primary human T cells specific to various EBV antigens (i.e. Epstein-Barr nuclear antigen 3A, 3B and BamHI-M leftward reading frame) via transfer of modified TCR genes that are either coupled to CD3zeta or Fc(epsilon)RIgamma. TCR-transduced T cells from 20-60% of donors (total number of 25) demonstrated specific lysis of EBV peptide-loaded target cells, whereas lymphoblastoid cell lines expressing native EBV antigens were not killed by any of the EBV-specific T cell populations. This non-responsiveness, confirmed at the level of nuclear factor of activated T cells activation, is not due to receptor configuration since identical receptor formats specific for melanoma antigens successfully re-targeted T cells to native melanoma cells. In an effort to generate a more potent receptor, we introduced a CD28 domain into one of the EBV-specific TCR. This TCR did not affect the cytotoxic response of re-targeted T cells, but dramatically enhanced antigen-specific IFNgamma production. We therefore conclude that these novel CD28-containing EBV-specific TCRs provide a basis for further development of TCR gene transfer to treat EBV-induced diseases.

OX40 Ligation of CD4+ T Cells Enhances Virus-Specific CD8+ T Cell Memory Responses Independently of IL-2 and CD4+ T Regulatory Cell Inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.

The clinical use of LMP2-specific cytotoxic T lymphocytes for the treatment of relapsed EBV +ve Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL)

EBV-associated HD and NHL show type II latency expressing subdominant EBV antigens such as LMP2, which may serve as targets for immunotherapy. Previously, we used polyclonal EBV-specific CTL in patients with relapsed EBV +ve HD and saw 2 CRs and 1 PR in 11 patients. We now hypothesize that CTL specifically targeting LMP2 might have greater efficacy. LMP2-CTL were generated using dendritic cells and lymphoblastoid cell lines which had been genetically modified to overexpress LMP2 by transduction with an Ad5f35LMP2 vector.