Epstein-Barr Virus-specific T-cell Research Articles

Constitutive Interleukin-7 Cytokine Signaling Enhances the Persistence of Epstein-Barr Virus-Specific T-Cells.

The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein-Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179).

Combining CD34+ stem cell selection with prophylactic pathogen and leukemia directed T-cell immunotherapy to simultaneously reduce graft versus host disease, infection, and leukemia recurrence after allogeneic stem cell transplant.

We designed a trial to simultaneously address the problems of graft versus host disease (GVHD), infection, and recurrence of malignancy after allogeneic stem cell transplantation. CD34+ stem cell isolation was used to minimize the development of acute and chronic GVHD. Two prophylactic infusions, one combining donor-derived cytomegalovirus, Epstein-Barr virus, and Aspergillus fumigatus specific T-cells and the other comprising donor-derived CD19 directed chimeric antigen receptor (CAR) bearing T-cells, were given 21-28 days after transplant. Two patients were transplanted for acute lymphoblastic leukemia from HLA identical siblings using standard doses of cyclophosphamide and total body irradiation without antilymphocyte globulin. Patients received no post-transplant immune suppression and were given no pre-CAR T-cell lymphodepletion. Neutrophil and platelet engraftment was prompt. Following adoptive T-cell infusions, there was rapid appearance of antigen-experienced CD8+ and to a lesser extent CD4+ T-cells. Tetramer-positive T-cells targeting CMV and EBV appeared rapidly after T-cell infusion and persisted for at least 1 year. CAR T-cell expansion occurred and persisted for up to 3months. T-cell receptor tracking confirmed the presence of product-derived T-cell clones in blood targeting all three pathogens. Both patients are alive over 3 years post-transplant without evidence of GVHD or disease recurrence. Combining robust donor T-cell depletion with directed T-cell adoptive immunotherapy targeting infectious and malignant antigens permits independent modulation of GVHD, infection, and disease recurrence. The combination may separate GVHD from the graft versus tumor effect, accelerate immune reconstitution, and improve transplant tolerability.

Rapid single-cell identification of Epstein–Barr virus-specific T-cell receptors for cellular therapy

Background and aimsEpstein–Barr virus (EBV) is associated with solid and hematopoietic malignancies. After allogeneic stem cell transplantation, EBV infection or reactivation represents a potentially life-threatening condition with no specific treatment available in clinical routine. In vitro expansion of naturally occurring EBV-specific T cells for adoptive transfer is time-consuming and influenced by the donor's T-cell receptor (TCR) repertoire and requires a specific memory compartment that is non-existent in seronegative individuals.The authors present highly efficient identification of EBV-specific TCRs that can be expressed on human T cells and recognize EBV-infected cells. Methods and ResultsMononuclear cells from six stem cell grafts were expanded in vitro with three HLA-B*35:01- or four HLA-A*02:01-presented peptides derived from six EBV proteins expressed during latent and lytic infection. Epitope-specific T cells expanded on average 42-fold and were single-cell-sorted and TCRαβ-sequenced. To confirm specificity, 11 HLA-B*35:01- and six HLA-A*02:01-restricted dominant TCRs were expressed on reporter cell lines, and 16 of 17 TCRs recognized their presumed target peptides. To confirm recognition of virus-infected cells and assess their value for adoptive therapy, three selected HLA-B*35:01- and four HLA-A*02:01-restricted TCRs were expressed on human peripheral blood lymphocytes. All TCR-transduced cells recognized EBV-infected lymphoblastoid cell lines. ConclusionsThe authors’ approach provides sets of EBV epitope-specific TCRs in two different HLA contexts. Resulting cellular products do not require EBV-seropositive donors, can be adjusted to cell subsets of choice with exactly defined proportions of target-specific T cells, can be tracked in vivo and will help to overcome unmet clinical needs in the treatment and prophylaxis of EBV reactivation and associated malignancies.

CD45RA expressing lymphocytes inhibit the outgrowth of Epstein-Barr Virus Specific T-cells from Patients with EBV+ Lymphoma

Abstract EBV antigen specific T-cells (EBVSTs) are difficult to expand from patients with EBV-positive lymphoma, perhaps because they circulate with low frequency or are rendered dysfunctional by the suppressive lymphoma microenvironment. As a result, we were unable to produce EBVSTs from ~25% of EBV+ lymphoma patients in our clinical trial (NCT01555892). To enrich memory T-cells prior to EBVST generation, we depleted the CD45RA+ subset of PBMCs, that includes naïve T-cells (TN), NK-cells, T-stem cells (TSCM) and terminally differentiated T-cells (TEMRA). CD45RA depletion (RAD) enhanced EBVST proliferation, antigen-specificity, polyfunctionality, cytotoxicity and tumor clearance in a murine model, and most importantly, enabled the outgrowth of EBVSTs from patient PBMCs that had previously failed to respond. This observation led to our hypothesis that bystander CD45RA+ cells inhibit the outgrowth of memory EBV antigen specific T-cells. TCR-β chain sequencing showed that the clonotypes with greatest expansion in RAD-EBVSTs failed to expand or expanded poorly in EBVSTs from whole PBMCs (W-EBSVTs), reflecting a more selective clonal expansion in RAD-EBVSTs. TCR repertoire of W-EBVSTs was more similar to that of EBVSTs derived from CD45RA+ PBMCs that lacked detectable EBV specificity and to that of the original PBMCs. Subsets add back to RAD-PBMCs suggested that the CD3+ CD45RA+ subset inhibits the reactivation and clonal expansion of EBV-Ag specific memory T-cells. This may represent a physiological strategy to prevent the oligoclonal outgrowth of inappropriate memory T-cells during primary infection. This approach has been translated into our ongoing clinical trials using NCT01555892 and NCT04288726 using EBVST against lymphoma. This work was funded and supported by CPRIT RP160283- Baylor College of Medicine Comprehensive Cancer Training Program, NIH-NCI P50 CA126752, American Society of Gene and Cell Therapy (ASGCT) Career Development Award, and Tessa Therapeutics.

Comprehensive profiling of ATA188, an off-the-shelf, allogeneic epstein-barr virus-specific T-cell immunotherapy for progressive multiple sclerosis

Comprehensive profiling of ATA188, an off-the-shelf, allogeneic epstein-barr virus-specific T-cell immunotherapy for progressive multiple sclerosis

Transcriptional Landscape and TCR Diversity of ATA188, a Pre-manufactured, Allogeneic Epstein-Barr Virus-specific T-cell Immunotherapy for Patients with Multiple Sclerosis (MS) (4397)

Transcriptional Landscape and TCR Diversity of ATA188, a Pre-manufactured, Allogeneic Epstein-Barr Virus-specific T-cell Immunotherapy for Patients with Multiple Sclerosis (MS) (4397)

Targeting CD39 in Cancer Reveals an Extracellular ATP- and Inflammasome-Driven Tumor Immunity.

We explored the mechanism of action of CD39 antibodies that inhibit ectoenzyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP-P2-mediated proinflammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP-P2X7-ASC-NALP3-inflammasome-IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Efficacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the significant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infiltration into T cell-poor tumors and rescued anti-PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and suppressed human B-cell lymphoma in the context of autologous Epstein-Barr virus-specific T-cell transfer. SIGNIFICANCE: Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP-P2X7-inflammasome-IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti-PD-1 resistance, and potentially enhances the efficacy of adoptive T-cell transfer.This article is highlighted in the In This Issue feature, p. 1631.

Non-uniform in vivo Expansion of Epstein-Barr Virus-Specific T-Cells Following Donor Lymphocyte Infusion for Post-transplant Lymphoproliferative Disease.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication of T-lymphocyte deplete allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with PTLD refractory to Rituximab, donor lymphocyte infusion (DLI) is established as a successful option for salvage therapy. However, although in vivo lymphocyte expansion has been correlated with good clinical outcome following DLI, the specificity and functional characteristics of EBV-specific T-cell responses remain poorly characterized. Here we describe two patients with Rituximab-refractory PTLD complicating T-cell deplete allo-HSCT, both of whom were successfully rescued with 1 × 106/Kg unselected stem cell donor-derived DLI. Prospective analyses revealed that complete clinical and radiological responses were associated with in vivo expansion of T and NK cells. Furthermore, EBV MHC tetramer, and interferon gamma analyses revealed a marked increase in EBV-specific T-cell frequency from 4 weeks after DLI. Reactivity was demonstrated against a range of EBV latent and lytic antigens, including those detected in tumor biopsy material. The immunodominant EBV-specific T cell response expanding in vivo following infusion matched the dominant response present in the DLI preparations prior to administration. Furthermore, differences in the repertoire of subdominant antigen-specific T-cells were also detected, suggesting that antigen-encounter in vivo can shape the immune response. These results demonstrate the value of prospectively studying in vivo T-cell responses, by facilitating the identification of important specificities required for clinical efficacy. Applying this approach on a larger scale promises to yield data which may be essential for the optimization of future adoptive immunotherapeutic strategies for PTLD.

High incidence of hematologic malignancy relapse after allogeneic transplantation in patients with low Epstein-Barr virus–specific T-cell counts

BackgroundAssociation between low counts of herpesvirus-specific T cells and subsequent relapse of hematologic malignancy has been shown in two retrospective studies. MethodsHere we present results of a prospective validation study. Multiple subsets of Epstein-Barr virus (EBV)–specific T cells were measured in 69 patients on day 56 and 84, using intracellular flow cytometry after incubation of blood mononuclear cells (MNCs) with EBV peptides or lysate. ResultsAll EBV T-cell subsets measured, both on day 56 and 84, were lower in patients who did versus did not subsequently relapse. This was most significant for day 56 EBV lysate-stimulated CD8 T cells producing interferon-gamma. Patients with day 56 counts of this subset >5/µL had a significantly lower likelihood of relapse compared with those with ≤5/µL (subhazard ratio, 5.7; P = 0.007). Similar significant associations were shown for a total of seven EBV T-cell subsets on day 56 and nine subsets on day 84. However, sensitivity and specificity of relapse prediction using the count of any subset was low (area under the curve of receiver-operator characteristic curve was <0.8). DiscussionIn conclusion, the association between EBV T-cell counts and subsequent relapse is valid. However, its clinical utility appears to be limited.

The pathology of epstein-barr virus lymphoproliferations.

The pathology of epstein-barr virus lymphoproliferations.

Modulating Interlukin-7 (IL-7) cytokine receptor signaling to enhance the persistence and anti-tumor efficacy of Epstein-Barr Virus (EBV) specific T-cells in EBV positive malignancies

Abstract EBV-specific T-cells (EBVSTs) targeting viral proteins in EBV+ lymphoma patients have produced 50% clinical response. Limited T-cell expansion and persistence attributed to lack of cytokines in the tumor microenvironment (TME) remains a major challenge. IL-7 signaling has survival and anti-apoptotic effects on T-cells, however, IL-7 availability in vivo is limited and the IL7 receptor is downregulated on activated T-cells. We aim to overcome this limitation of suppressive TME by augmenting IL-7 signaling. We recently reported the transgenic expression of a constitutively active IL-7 receptor (C7R) to provide the proliferative and survival advantage in T-cells. Hence, we hypothesize that EBVSTs endowed with cytokine-independent C7R signaling will have resistance to the immunosuppressive TME and increased persistence and anti-tumor potency. C7R expressing EBVSTs demonstrated enhanced antigen-specific IFN-γ release and superior target specific cytotoxicity in-vitro. In contrast to unmodified EBVSTs, C7R-EBVSTs demonstrated EBV antigen dependent, cytokine-independent proliferation and survival, associated with increased phosphorylated STAT5. Furthermore, C7R signaling provided resistance against TGFβ, Arginase-I, and M2 macrophages, as demonstrated by increased proliferation attributed to decreased apoptosis in C7R-EBVSTs. Following adoptive transfer in an EBV+ murine xenograft model, C7R-EBVSTs persisted significantly longer and demonstrated tumor clearance, while unmodified EBVSTs failed. C7R signaling augments EBVSTs potency and persistence against suppressive components of TME in our preclinical models. Once effective, the findings will be translated to clinical trials in EBV+ malignancies.

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.

Normalizing ELISPOT to quantify human cytomegalovirus (HCMV) and Epstein Barr-virus (EBV) specific T-cell response in kidney transplant recipients

Normalizing ELISPOT to quantify human cytomegalovirus (HCMV) and Epstein Barr-virus (EBV) specific T-cell response in kidney transplant recipients

Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines.

Epstein-Barr virus-related post-transplant lymphoproliferative disorders are recognized as a significant cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. To better define current understanding of post-transplant lymphoproliferative disorders in stem cell transplant patients, and to improve its diagnosis and management, a working group of the Sixth European Conference on Infections in Leukemia 2015 reviewed the literature, graded the available quality of evidence, and developed evidence-based recommendations for diagnosis, prevention, prophylaxis and therapy of post-transplant lymphoproliferative disorders exclusively in the stem cell transplant setting. The key elements in diagnosis include non-invasive and invasive methods. The former are based on quantitative viral load measurement and imaging with positron emission tomography; the latter with tissue biopsy for histopathology and detection of Epstein-Barr virus. The diagnosis of post-transplant lymphoproliferative disorder can be established on a proven or probable level. Therapeutic strategies include prophylaxis, preemptive therapy and targeted therapy. Rituximab, reduction of immunosuppression and Epstein-Barr virus-specific cytotoxic T-cell therapy are recommended as first-line therapy, whilst unselected donor lymphocyte infusions or chemotherapy are options as second-line therapy; other methods including antiviral drugs are discouraged.

Abstract B17: Banked Epstein-Barr virus specific T-cells for treatment of EBV+ lymphoma

Abstract Standard intensive chemotherapy and hematopoietic stem cell transplant usually benefit patients with Hodgkin (HL) and Non-Hodgkin Lymphoma (NHL), but for those with T or NK-T NHL or who relapse, the prognosis is less favorable. Furthermore, substantially increased morbidity and mortality from the complications of aggressive chemotherapies is evident for many years, even in patients who are cured of their original disease. About 30% of all lymphomas carry the Epstein-Barr virus (EBV) genome and express 4 viral proteins (EBNA1, LMP1, LMP2 ± BARF1), a pattern termed Type 2 latency. T-cells specific for these antigens circulate with low frequency in healthy donors, but are rendered anergic in patients by their immunosuppressive tumors. We have previously shown that potent in vitro T-cell stimulation can reverse anergy. Virus-specific T-cells specific for the EBV antigens (EBVSTs) have produced complete remissions in 60% of patients with multiply relapsed EBV+ HL and NHL. Though successful, our earlier method was labor intensive and prolonged, and the requirement for live EBV and adenoviral (Ad) vectors increased costs and regulatory complexity. We have overcome these problems by replacing Ad vectors with peptide mixtures (pepmixes) and the EBV lymphoblastoid cell lines (LCL) with professional antigen presenting cells to provide costimulation. We therefore seek to produce a safe and effective immunotherapy for relapsed, refractory EBV+ lymphoma that can be implemented rapidly by creating and characterizing a bank of T-cells targeted to the four EBV antigens expressed in EBV+ HL and NHL. Thus far, we have successfully screened 30 eligible blood donors and manufactured several EBVST lines with specificity for the four Type 2 EBV latency proteins using the rapid pep-mix method. This strategy robustly and rapidly produces higher frequencies of EBV-specific T cells that kill LCLs that naturally express viral antigens, showing that the pepmixes are not recruiting low-avidity T-cells. T-cell lines will be selected and infused based on their ability to recognize EBV antigens through HLA alleles shared with the recipient. The in vivo expansion and persistence of the infused EBVSTs will be evaluated together with their ability to reactivate endogenous T-cells specific for non-viral tumor antigens. We hypothesize that allogeneic, partially HLA-matched T-cells can produce benefit in patients with lymphoma both by direct tumor cell killing and by creating a proinflammatory tumor microenvironment that reactivates and restores effective endogenous tumor-specific T-cells. Citation Format: Rayne H. Rouce, Serena K. Perna, Gayatri Vyas, Sandhya Sharma, Natalia Lapteva, Ann Leen, Cliona M. Rooney, Helen E. Heslop. Banked Epstein-Barr virus specific T-cells for treatment of EBV+ lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B17.

First-in-Man Clinical Results With Good Manufacturing Practice (GMP)-compliant Polypeptide-expanded Adenovirus-specific T Cells After Haploidentical Hematopoietic Stem Cell Transplantation

Adoptive immunotherapy against viral infections is a promising treatment option for patients after hematopoietic stem cell transplantation. However, the generation of virus-specific T cells is either cost-intensive or time-consuming. We developed the first GMP-compliant protocol to generate donor-derived adenovirus (HAdV), cytomegalovirus, and Epstein-Barr virus-specific T-cell lines (TCLs) within 12 days by the use of overlapping polypeptides derived from different viruses in combination with IL-15. Two patients after undergoing haploidentical hematopoietic stem cell transplantation with HAdV viremia displaying rising viral loads despite treatment with cidofovir received 1×10 donor-derived short-term expanded HAdV-specific TCLs per kg body weight. In both patients, HAdV-specific T cells could be detected by IFN-γ-ELISpot 30 and 22 days postinfusion, and resulted in complete clearance or >1.5 log reduction of viral load within 15 and 18 days, respectively. This protocol facilitates rapid and cost-effective generation of virus-specific TCLs, which appear to provide an effective treatment option.

Upregulation of CXCR3 expression on CD8+ T cells due to the pervasive influence of chronic hepatitis B and C virus infection

Chronic systemic 'latent' viral infections such as Cytomegalovirus infection (CMV) are known to leave a fingerprint in the total T-cell population. We investigated whether chronic infections with a 'persistent' viremia, such as chronic hepatitis B and C (CHB, CHC), characterized by local organ-specific inflammation, also impact the total peripheral T-cell population or other virus specific T-cells that do not target hepatitis viruses. No phenotypic or functional differences were found between CD8(+) T-cells or CMV- or Epstein-Barr virus specific T-cells in viral hepatitis and healthy controls (HC). However, expression of chemokine-receptor CXCR3 was significantly higher on total peripheral CD8(+) T-cells of CHB or CHC patients compared to HC (p<0.005) which may reflect the pervasive influence of a persistent viral infection, even when restricted to the liver. In CHB higher CXCR3 expression was associated with positive HBeAg-status and correlated with the percentage of HBsAg expressing hepatocytes found in liver biopsies, both pointing to a relation between CXCR3 expression and disease activity. In fact chemokine-receptors such as CXCR3 are important for T-cell recruitment to the liver and chemokine-ligands specific for CXCR3 are upregulated in chronic hepatitis. Modulating chemokine(receptor) expression could be a potential target for future therapy to optimize the anti-viral immunologic environment in the liver.

High Epstein–Barr virus-specific T-cell counts are associated with near-zero likelihood of acute myeloid leukemia relapse after hematopoietic cell transplantation

High Epstein–Barr virus-specific T-cell counts are associated with near-zero likelihood of acute myeloid leukemia relapse after hematopoietic cell transplantation

Latent CMV And EBV Infections And The Mobilization Of Viral-specific And Senescent T-cells With Exercise

Reactivating latent viruses cause senescent T-cells to accumulate within the so-called "immune space", reducing the naïve T-cell repertoire and increasing host infection risk. Although senescent T-cells (i.e. KLRG1+/CD57+) are mobilised into the blood by acute exercise, the impact of latent cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections on this response is unknown. It remains to be determined also if the mobilized cells react with epitopes of the CMV or EBV virus in those infected. PURPOSE: To determine: (1) if CMV and EBV specific T-cells are mobilised into blood in response to acute exercise; and (2) the impact of latent CMV and EBV infections on the exercise-responsiveness of senescent T-cells. METHODS: Twenty-four healthy males (age 28.5 ± 5) completed a 30-min cycling protocol at 85% of maximum power. Mononuclear cells were isolated from blood before, immediately after and 1hour after exercise and labelled with MHC class I Pentamers specific to CMV (pp65) or EBV (LMP-2) and anti CD3 and CD8 antibodies. Naïve (KLRG1-/CD28+/CD57-), effector-memory (KLRG1+/CD28+/CD57-) and senescent (KLRG1+/CD28-/CD57+) phenotypes were determined on pentamer positive and negative CD8+ T-cells by flow cytometry. ELISA tests were used to determine CMV and EBV serostatus. RESULTS: Exercise increased the numbers of CMV and EBV-specific T-cells by 158% and 200% respectively (p<.01), and altered the proportions of senescent (+44%) and naïve (-20%) CD8+ T-cells (p<.05). CMV seropositive (n = 10) had a greater (+136%; p<.01) proportion of senescent and a lower (-33%; p<.01) proportion of naïve CD8+ T-cells compared to seronegative (n= 14), and demonstrated a blunted senescent T-cell response to exercise (+38% vs +51%; p<.01). Senescent markers on both CMV and EBV-specific T-cells increased with exercise (+53% and +76% respectively; p<.05). EBV serostatus did not influence the proportions or exercise response of the CD8+ T-cell populations (p>.05). CONCLUSION: CD8+ T-cells specific to CMV and EBV are mobilized into blood with acute exercise; however, the relative exercise responsiveness of senescent T-cells is blunted with CMV infection. Future work should determine the fate of the mobilized senescent cells and the potential impact that exercise might have on creating "immune space" in those infected with CMV.

Epstein Barr Virus (EBV) Reactivation After Reduced Intensity Conditioning (RIC) Unrelated Umbilical Cord Blood Transplantation (UCBT)

AbstractAbstract 2334Umbilical cord blood has been increasingly used as an alternative stem cell source for allogeneic hematopoietic stem cell transplantation (allo-HSCT). RIC regimens have been increasingly used prior to allo-HSCT with the aim to decrease transplant-related mortality (TRM) in patients ineligible for standard conditioning. Because of the slow kinetics of immune reconstitution after UCBT, previous studies showed that EBV reactivation and EBV induced lymphoprolipherative disease (LPD) may be of matter of concern, particularly in the reduced intensity setting. The aim of this analysis was to investigate the features of EBV reactivation in 33 patients who underwent RIC UCBT between January 2005 and June 2009.In our series, 30 patients (91%) received a double UCBT and 3 patients (9%) received a single UCBT, with a median of 4.0×107/Kg (range, 2.2–5.8) total nucleated cells. Donors and recipients were mismatched with one mismatch in 14 (43%) of cases and 2 mismatches in 19 (57%) of cases. A RIC including fludarabine, cyclophosphamide and low dose TBI was used in 29 cases (88%), while 8 patients (24%), who were not heavily pretreated before UCBT, received ATG. EBV reactivation was defined as any EBV PCR load above 1000 copies of EBV DNA /105 cells. EBV LPD was defined as biopsy or autopsy proven post-transplantation lymphoma, or reactivation along with computerized tomography nodal or soft-tissue abnormalities consistent with LPD. Patients with EBV viral load >1000 copies/105 cells on at least two consecutive occasions were treated with rituximab at a dose of 375 mg/m2 weekly until clearance of EBV viremia.Engraftment occurred in 25 patients (76%) at a median time of 12 days after UCBT (range, 8–60). Clinically significant grade II to IV acute GVHD occurred in 5 of cases (15%). The cumulative incidence of EBV reactivation at 6 months and three years after allo-HSCT was 13% and 17%. EBV reactivation was observed at a median of 132 (range 85–438) days after allo-HSCT, with 3 (60%) reactivations occurring during the first 6 months. In 28 patients (85%), the EBV load remained less than 1000 EBV copies/105 cells at all time, and none of these patients experienced any sign or symptom of LPD. The remaining 5 patients (15%) experienced at least one EBV reactivation episode. Among the 5 patients experiencing EBV reactivation, 2 patients received ATG as part of their RIC. Among these 5 patients, 1 patient experienced an EBV load superior to 1000/105 cells at a single time point after allo-HSCT. In this patient, there were no concomitant clinical symptoms and the EBV load normalized spontaneously. Only this patient had a normal T-lymphocyte count at the time of reactivation. Four patients who had EBV DNA levels exceeding 1000 copies/105 cells on 2 or more occasions were treated with a median of 3 (range, 1–8) rituximab infusions. Two patients responded to rituximab, but 2 patients developed LPD (cumulative incidence of 6 % at three years). Both of our patients were severely immunosupressed with high dose corticosteroid therapy at the time of the occurrence of EBV LPD. One of these 2 patients died before receiving any other anti-EBV therapy. In the other patient, LPD could be controlled after additional chemotherapy, radiotherapy and 2 infusions of EBV specific Cytotoxic T-cell Lines (CTLs). Three of five patients (60%) who experienced EBV reactivation had Human Herpes Virus 6 (HHV 6) detected in the same blood sample by PCR. With a median follow-up of 468 (range, 92–1277) days post allo-HSCT among surviving patients, 21 patients (64%) were still alive and the overall survival (OS) was 62% at 3 years. 5 patients died of disease progression and 7 patients died of transplant-related complications. One patient died of LPD. There was no statistically significant difference in terms of OS or TRM between those patients who experienced an EBV reactivation after UCBT and those who did not (OS: log rank test, p=0.33, TRM: Gray test, p= 0.82). Univariate analysis did not find any risk factors significantly different between subgroups with and without EBV reactivation.Overall, this study shows the rate of EBV reactivation after RIC UCBT to be comparable to the incidence expected with RIC mismatched unrelated bone marrow or peripheral blood HSCT. Despite small numbers, our observations support close EBV monitoring and the use of pre-emptive rituximab treatment since some cases may progress to LPD requiring additional interventions such as EBV-specific CTLs. Disclosures:No relevant conflicts of interest to declare.