Cytotoxic T Lymphocytes Lines Research Articles

Complete Tumor Responses in Lymphoma Patients Receiving Autologous Cytotoxic T Lymphocytes Targeting Epstein Barr Virus (EBV) - Latent Membrane Proteins

Abstract 956The tumor cells of EBV-associated Hodgkin’s Lymphoma (HL) and of some non-Hodgkins lymphoma (NHL) express the subdominant EBV antigens LMP1 and LMP2 (Type II viral latency), and these may serve as target antigens for immunotherapy. We hypothesized that cytotoxic T lymphocytes (CTLs) enriched for effector cells specifically targeting LMP antigens would have efficacy in patients with EBV+ lymphoma. We prepared antigen presenting cells (dendritic cells for the initial stimulation, EBV- lymphoblastoid cell lines (LCLs) for the subsequent) that expressed transgenic and inactive LMP1 (ΔLMP1) and/or LMP2, following transduction with the adenoviral vectors Ad5f35LMP2 (n=16) or Ad5f35ΔLMP1-I-LMP2 (n=27). All LMP-CTL lines were polyclonal as measured by flow cytometry CD4+ (mean 17+/−18%; range 1–92%) and CD8+ (mean 74 +/− 25%; range 1–99%) T-cells. Analysis of memory markers revealed mixed populations of CD45RA- CD62L- T-cells (mean 45+/−15%; range 31–63%) and CD45RA- CD62L+ T-cells (mean 34+/−5%; range 28–41%). CTL lines had specificity for CD4+ and CD8+ restricted LMP2 epitopes alone (median 1 epitope; range 0–7) or both LMP1 and LMP2 epitopes (median 3 epitopes; range 0–9) per CTL line, as determined using overlapping LMP1 and LMP2 peptide pools to stimulate the T cells in γ-IFN ELISPOT assays. 43 patients with EBV+ HL (n= 18) and NHL (n=25) have been treated and received between 4×107/m2 and 3.2×108/m2 T cells; 16 received LMP2 CTLs and 27 were given LMP1/2 CTLs. No immediate toxicity was observed. Within 1 to 4 weeks after CTL infusion, the numbers of LMP-specific T-cells in peripheral blood rose 2 to 70 fold, and elevated levels persisted for up to 3 months. Lymph node biopsies from 3 patients taken 3–6 months post CTL showed selective accumulation of LMP-specific T-cells in lymph nodes compared to peripheral blood. 21/22 high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment remain in remission for a median of 2.5 years after CTL with a PFS of 80% at 3 years. 21 patients had detectable disease at the time of CTL infusion. Following treatment, 4 had progressive disease by 8 weeks, 1 is too early and 16 had clinical responses. The median duration of the clinical responses is 1.5 years with 1 stable disease, 3 partial responses and 13 complete responses. The 3 year progression free survival for patients treated with disease is 60%. We compared outcomes between patients who received LMP2 CTL alone versus LMP1 and 2-specific CTL. For high-risk patients treated with CTLs as adjuvant, the 3 year PFS is 70% for LMP2 CTL recipients versus 100% for recipients of LMP1/2-CTLs (p=0.06). The 3 year PFS in relapsed patients receiving LMP2-CTL was 50% versus 62% for recipients of LMP1/2 CTL (p=0.3). In conclusion, immunotherapy with CTLs targeting LMP antigens is well tolerated in patients with EBV+ lymphoma. Infused LMP-CTL accumulate at tumor sites and have induced complete and sustained clinical responses in 13/20 patients with relapsed disease. We are now implementing a simpler LMP-specific CTL expansion protocol to enable a definitive efficacy study. Disclosures:Pro:Allos Therapeutics: Honoraria.

Improving T Cell Therapy for Relapsed EBV-Negative Hodgkin's Lymphoma by Targeting Upregulated MAGE-A4

Abstract 1658Patients with Hodgkin’s Lymphoma (HL) who relapse after hematopoietic stem cell transplant (HSCT) have limited options for long-term cure. We have shown that infusion of cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV+ HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative strategy is to target the cancer/testis antigen MAGE-A4 which is present in EBV antigen-negative Hodgkin’s lymphoma tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors. We explored the feasibility of combining adoptive T-cell therapy with epigenetic modification of tumor antigen expression. We first verified whether MAGE-A4-specific CTL could be expanded from autologous and allogeneic donors. Dendritic cells from healthy donors (n=15) or HL patients (n=9) were pulsed with overlapping peptides spanning the MAGE-A4 protein. Healthy donor T-cell lines expanded to sufficient numbers for clinical use and predominantly comprised CD8+ cytotoxic T cells (mean 60%; range 42–81%) and CD4+ T helper cells (mean 20%; range 8–45%). A mean response of 152 interferon gamma (IFNγ) spot forming cells (SFC)/1 × 105 cells (median 78; range 12–635) was observed against MAGE-A4 peptides compared to a mean of 6 IFNγ SFC/1×105 cells (median 4; range 0–30) to irrelevant peptides. Both CD4+ and CD8+ T cell subsets contributed to the interferon response. In T cells expanded from the peripheral blood of patients with HL, a mean of 296 SFC/1×105 cells (range 32–1031) responded to MAGE-A4 peptides compared to a mean of 5 SFC/1×105 cells (range 1–11) responding to irrelevant targets. Five of fifteen CTL lines generated from healthy donors showed specificity for a particular peptide (aa266-285, NPARYEFLWGPRALAETSYV). In contrast, T-cell lines derived from HL patients showed a range of MAGE-A4 epitope specificities. Peripheral blood mononuclear cell and dendritic cell numbers were reduced, and T cells from these patients failed to expand after several stimulations consistent with the profound immune deficiency frequently seen in HL patients. We next evaluated the effect of combining MAGE-CTL with the epigenetic-modifying demethylating agent 2’-deoxy-5’azacytidine. To determine if decitabine would negatively affect the function of the TAA-specific T cells, we examined the effects of the drug on the function of MAGEA4-specific T cells in vitro. Our results showed that treatment of T cells with decitabine – in concentrations that exceed expected levels following drug treatment (1 μM) - did not adversely affect the phenotype and function of MAGE-A4 specific T cells in vitro. To evaluate the in vivo effects of decitabine on tumor antigen specific T cells, patient samples were obtained before and after treatment. In patients who received a decitabine containing regimen (75 mg/m2 daily × 5 days) (n=2), one of whom had clinical responses on PET scan, we observed that MAGE-specific T cells were of greater magnitude and recognized a broader number of MAGE-A4 epitopes after decitabine treatment (see Figure below – a and b are patients with a decitabine containing regimen; c and d are patients receiving no decitabine). In patients (n= 2) who did not receive a decitabine containing regimen, the magnitude of MAGE-specific reactivity was reduced, as measured by IFNγ Elispot. These results suggest that although decitabine may modulate immune function, the effect of other chemotherapies may result in the lack of appreciable endogenous T-cell responses to tumor antigens in these patients. Hence, adoptive transfer of MAGE-A4 specific T cells combined with the administration of epigenetic-modifying drugs to increase expression of the target antigens may improve the efficacy of treatment of relapsed Hodgkin’s Lymphoma. [Display omitted] [Display omitted] Disclosures:Younes:Celgene: Speakers Bureau; Syndax: Research Funding; SBIO: Research Funding; Genentech: Research Funding; Sanofi-Aventis: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau, Travel Expenses; Gilead: Honoraria; Pharmacyclic: Honoraria.

Neurons are MHC Class I-Dependent Targets for CD8 T Cells upon Neurotropic Viral Infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage.

Identification of HLA-A24-restricted CD8+ cytotoxic T-cell epitopes derived from mammaglobin-A, a human breast cancer–associated antigen

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8(+) cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8(+) CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8(+) CTL lines lysed the HLA-A24(+)/Mam-A(+) stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24(-)/Mam-A(+) and HLA-A24(+)/Mam-A(-) breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8(+) CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.

Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation.

Hematopoietic stem cell transplant (HSCT) is the treatment of choice for a proportion of patients with hematologic malignancies as well as for non-malignant diseases. However, viral infections, particularly Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus (Ad), remain problematic after transplant despite the use of antiviral drugs. We have shown that cytotoxic T lymphocytes (CTL) generated against CMV-pp65, EBV and Ad antigens in a single culture are capable of controlling infections with all three viruses after HSCT. Although pp65-specific CTL have proved efficacious for the control of CMV infection, several reports highlight the importance of targeting additional CMV antigens. To expand multivirus-specific T cells with activity against both CMV-pp65 and CMV-IE-1, peripheral blood mononuclear cells (PBMC) were transduced with the adenoviral vector (Ad5f35-IE-1-I-pp65). After 9-12 days the CTL were restimulated with autologous EBV-transformed B cells transduced with the same Ad vector. After 18 days in culture nine CTL lines expanded from less than 1.5 × 10(7) PBMC to a mean of 6.1 × 10(7) T cells that recognized CMV antigens pp65 [median 273 spot-forming cells (SFC), range 47-995] and IE-1 (median 154 SFC, range 11-505), the Ad antigens hexon (median 153 SFC, range 26-465) and penton (median 37 SFC, range 1-353), as well as EBV lymphoblastoid cell lines (median 55 SFC, range 9-301). Importantly, the T cells recognized at least two antigens per virus and lysed virus peptide-pulsed targets. CTL that target at least two antigens each of CMV, EBV and Ad should have clinical benefit with broad coverage of all three viruses and enhanced control of CMV infections compared with current protocols.

T cells expressing the activating NK-cell receptors KIR2DS4, NKG2C and NKG2D are elevated in paroxysmal nocturnal hemoglobinuria and cytotoxic toward hematopoietic progenitor cell lines

To investigate the presence of T cells with natural killer cell receptors (NKR) in paroxysmal nocturnal hemoglobinuria (PNH), and their potential involvement in clonal expansion of glycosylphosphatidylinositol (GPI)-deficient hematopoietic stem cells by selective immune attack to normal and not GPI-deficient hematopoietic stem cells. By flow cytometry, the frequency and number of T cells expressing NKR was evaluated in 39 PNH patients and compared to healthy controls. Elevated T cell subsets in PNH were assessed for differential cytotoxic lysis of GPI(+) and GPI(-) CD34(+) hematopoietic progenitor cell lines. In PNH patients, the frequency (p < 0.001) and absolute number of T cells expressing the NKR CD56 (p = 0.01) were significantly increased. Furthermore, a higher percentage of T cells expressed the activating NKR NKG2D (p < 0.01), NKG2C (p < 0.01), and KIR2DS4 (p = 0.01). Further characterization showed that these populations predominantly consist of CD8(+) effector memory CD45RA(+) T cells (T(EMRA)). NKR(+) cytotoxic T-lymphocyte lines isolated from PNH patient peripheral blood and bone marrow displayed high cytotoxicity towards CD34(+) hematopoietic progenitor cell lines and K562 cells, suggesting major histocompatibility complex class I-independent cytotoxicity. These cytotoxic T lymphocyte (CTL) lines are capable of differential lysis of GPI(+) and GPI(-) hematopoietic cell lines, however, not in all cases. This suggests that multiple factors, such as the highly activated status of in vitro cultured CTLs, influence whether GPI-dependent lysis occurs. The increased frequency of CD8(+) effector-memory T cells with activating NKR and cytotoxicity toward hematopoietic cell lines suggests involvement in bone marrow failure and clonal expansion in PNH.

High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells

AbstractThe cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell–mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02–restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME+ hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming–inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME+ hematologic malignancies.

新たなヒトT細胞株 &amp;ldquo;HOZOT&amp;rdquo; の発見とその免疫制御機構

新たなヒトT細胞株 &amp;ldquo;HOZOT&amp;rdquo; の発見とその免疫制御機構

Allogeneic virus-specific T cells with HLA alloreactivity do not produce GVHD in human subjects

Adoptive transfer of viral antigen-specific memory T cells can reconstitute antiviral immunity, but in a recent report a majority of virus-specific cytotoxic T-lymphocyte (CTL) lines showed in vitro cross-reactivity against allo-human leukocyte antigen (HLA) molecules as measured by interferon-γ secretion. We therefore reviewed our clinical experience with adoptive transfer of allogeneic hematopoietic stem cell transplantation donor-derived virus-specific CTLs in 153 recipients, including 73 instances where there was an HLA mismatch. There was no de novo acute graft-versus-host disease after infusion, and incidence of graft-versus-host disease reactivation was low and not significantly different in recipients of matched or mismatched CTL. However, we found that virus-specific T cell lines recognized up to 10% of a panel of 44 HLA disparate targets, indicating that virus-specific T cells can have cross-reactivity with HLA-mismatched targets in vitro. These data indicate that the adoptive transfer of partially HLA-mismatched virus-specific CTL is safe despite in vitro recognition of recipient HLA molecules.

Most Closely HLA-Matched Allogeneic Virus Specific Cytotoxic T-Lymphocytes (CTL) to Treat Persistent Reactivation or Infection with Adenovirus, CMV and EBV After Hemopoietic Stem Cell Transplantation (HSCT)

AbstractAbstract 829Adoptive transfer of donor-derived cytotoxic T lymphocytes (CTLs) can reconstitute antiviral immunity to Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus in recipients of allogeneic hemopoietic stem cell transplantation (HSCT). However, the time taken to prepare patient-specific products and the lack of virus-specific memory T cells in cord blood and seronegative donors restricts application. One means of avoiding growing CTLs for individual patients is to bank lines that are then available as an “off the shelf” product of most closely HLA-matched allogeneic cytotoxic T lymphocyte lines (CHM-CTLs). We are evaluating this strategy in a multicenter study through the NHLBI Specialized Centers for Cell-Based Therapy (SCCT) program. Our goal is to determine the feasibility, safety and antiviral activity of 3rd party CHM-CTLs in HSCT recipients who have viral reactivation or infection refractory to standard therapy. Using the NHLBI Production Assistance for Cellular Therapies (PACT) program, we manufactured and safety tested more than 30 multivirus CTL lines from normal donors by stimulating peripheral blood mononuclear cells with autologous monocytes and EBV-transformed LCLs, both transduced with an Ad5f35 vector encoding the CMV-derived pp65 antigen. All the lines are polyclonal comprising CD4+ (median 10%, range 1.5–99%) and CD8+ (median 83%, range 0–96%) T cells. These CTL lines were specific for CMV, EBV and adenovirus (mean 1463, 101, and 227 SFC/1×105 cells, respectively), with recognition of both CD8+ and CD4+ epitopes as determined using individual epitope peptides and overlapping Hexon and pp65 peptide pools (median 1.5; range 0–5 and median 3; range 0–10 pools/peptides recognized, respectively) in ELISPOT assays. These CHM-CTLs were available for administration to subjects who had persistent viral infection and matched at least one HLA antigen with a dose of up to 2.0 x107CHM-CTL/m2. We selected the CTL line for administration based on their specificity for the target virus through a shared allele, and on the overall level of HLA match. Patients were eligible for a subsequent infusion in the event of a partial response. To date 47 patients have been screened after marrow (n=9) blood (n=18) or cord (10 single, 10 double) transplantation and a potential line identified on 43. Of 4 patients with no line available 3 died of progressive adenovirus infection and one with CMV infection is alive. Of 43 patients with an identified line, 25 received lines that matched at 1 to 3 HLA antigens as either one (n=19), two (n=4) or 3 (n=2) CTL infusions. Five patients received CHM-CTLs for refractory EBV PTLD (Post Transplant Lymphoproliferative Disease), 11 patients for persistent CMV infection, 8 patients for persistent adenovirus infection and one for both CMV and adenovirus. There were no immediate adverse effects related to infusion, and the incidence of GVHD was low, with two patient having reactivation of previous acute skin or chronic GVHD, and two having transient skin rashes. Two patients developed transplant-associated microangiopathy but had other risk factors including Rapamycin use. Preliminary responses for the overall cumulative incidence of first CR/PR in the first 22 patients based on viral load by day 42 post-infusion is 77.3% (90% for CMV, 80% for EBV and 71% for adenovirus). Two patients with widespread EBV-PTLD (one CD20-negative and one refractory to Rituximab) achieved sustained complete responses by RECIST criteria. Of note complete antiviral responses occurred even in recipients of lines matching at only a single HLA antigen. For example, one patient with both CMV and adenovirus infections received a CTL line that only matched at HLA B53 and cleared both viruses and we were able to show specificity against both viruses through this one allele. Despite objective antiviral activity, immune monitoring studies using IFN-g ELISPOT assays showed little or no rise in peripheral blood virus-reactive T cells post-infusion, a result that contrasts with the large rise seen in recipients of donor-derived T cell lines. Nevertheless these preliminary results show the feasibility of adoptive immunotherapy for CMV, EBV, and adenovirus using banked allogeneic multivirus-CTLs, and that the approach may be applicable to a majority of recipients in need. The antiviral responses we observe are encouraging and suggest than an “off-the-shelf” CTL cell line may be a practical strategy. Disclosures:Off Label Use: T cell products manufactured under INDs.

T Cells Expressing the Activating NK Cell Receptors KIR2DS4, NKG2C and NKG2D Are Elevated In Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Cytotoxic towards Hematopoietic Progenitor Cell Lines

AbstractAbstract 2235 Background:Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease characterized by hemolysis due to an acquired mutation in the X-linked PIG-A gene in the hematopoietic stem cell (HSC). This leads to a clone of hematopoietic cells with deficient expression of glycosyl phosphatidyl inositol (GPI) anchored proteins at the cell membrane. The clinical evolution of PNH arises through clonal expansion of PIG-A mutated HSC which is insufficiently explained by the PIG-A mutation alone. Hypothetically, clonal expansion could result from autoreactive T cells selectively attacking normal HSC, whereas GPI deficient HSC are unharmed. Methods and Results:we investigated the presence of potentially autoreactive T cells in peripheral blood of patients with PNH (n = 39) by flow cytometry. We compared T cell subset frequencies and absolute numbers with healthy controls (n = 25) using Mann-Whitney U test. In PNH patients, T cells expressing the NK cell marker CD56 were significantly elevated, both in percentage (p < 0.001) and in absolute numbers (p < 0.01). Furthermore, the frequency of T cells expressing the activating NK cell receptors (NKRs) NKG2D (p < 0.01), NKG2C (p < 0.01), and KIR2DS4 (p = 0.01) was significantly increased. KIR2DS4+, NKG2C+ and NKG2D+ T cells mainly consist of highly differentiated effector memory CD45RA+ T cells (TEMRA) (KIR2DS4: median 90%, range 70–96%, NKG2C: median 83%, range 49–99%, NKG2D: median 40%, range 38–66%). A highly variable proportion of these T cell populations consists of γδ T cells (KIR2DS4: median 28%, range 6–72%, NKG2C: median 36%, range 3–75%, NKG2D: median 11%, range 7–40%). By 10 color flow cytometry, we examined NKR coexpression patterns. KIR2DS4+ and NKG2C+ T cells mainly coexpress either only NKG2D (KIR2DS4+ T cells: median 24%, range 2 – 74%, NKG2C+ T cells: median 21%, range 3–71%), or a combination of NKG2D, NKG2C and CD158b1/b2,j, but not inhibitory NKG2A (KIR2DS4+ T cells: median 16%, range 1 – 55%, NKG2C+ T cells: median 20%, range 1–60%). In contrast, NKG2D+ T cells generally do not express any other NKRs tested (median 77%, range 53–84%). NKG2D+ KIR2DS4+ cytotoxic T lymphocyte (CTL) lines isolated from PNH patient peripheral blood and bone marrow display high cytolytic activity towards CD34+ hematopoietic progenitor cell lines and MHC class I deficient K562 cells, suggesting T cell receptor independent cytolytic activity. These NKR+ CTL lines are capable of differentially lysing GPI+ and GPI- hematopoietic cell lines, however not in all cell line models and CTL lines. This suggests that multiple factors, as for example the highly activated status of in vitro cultured CTLs, influence whether or not GPI dependent lysis occurs. Conclusion:The increased frequency of T cells expressing activating NK cell receptors KIR2DS4, NKG2C, and NKG2D, with a CD8+ effector-memory phenotype and differences in cytotoxicity towards GPI+ and GPI- hematopoietic cell lines suggests that these T cell populations may be involved in bone marrow failure and expansion of PNH clones. Disclosures:Muus: Alexion: member of advisory board.

Allogeneic Virus-Specific T Cells with HLA Alloreactivity Do Not Produce Graft-Versus-Host Disease In Human Subjects

AbstractAbstract 1252We and others have recently established that T cell reactivity with non-self HLA (HLA alloreactivity) arises not only from naive T cells but also from the antigen-experienced T cell pool, including Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific T cells. Virus-specific T cells could therefore mediate graft-versus-host disease (GvHD) if infused into partially HLA mismatched recipients. We reviewed our clinical experience with adoptive transfer of allogeneic hematopoietic stem cell transplant donor-derived virus-specific T cell lines in 153 recipients who received donor-derived EBV-specific CTLs (N=114), bivirus CTLs specific for adenovirus and EBV (n=14), and trivirus CTLs specific for CMV, adenovirus and EBV (n=25). Seventy three donor-recipient pairs were partially HLA-mismatched, with the degree of HLA mismatching varying from one allele to a full haplotype. De novo GvHD did not develop after infusion of cytotoxic T lymphocytes (CTL), and the incidence of GvHD reactivation was 6.5% and not significantly different between recipients of HLA matched or mismatched CTL. Thus, virus-specific CTL did not mediate GvHD, even in recipients of partially matched CTL. Next we analyzed the HLA alloreactivity of four donor-infused bivirus-specific T cell lines, using activated T cells, that are known to lack CMV and EBV antigen expression, as antigen presenting cells (TAPC). We used a panel of 44 TAPC covering the most frequent HLA class I and II alleles. The CTL lines were labeled with CFSE and stimulated with TAPC for 6 hours, after which production of TNFα and IFNγ/IL-2 by CD4+ and CD8+ T cells in the CFSE-positive fraction was analyzed by flow cytometry. All CTLs responded to a number of TAPC, with some APC being recognized strongly. The majority elicited only weak or no response from the CTLs. We then assessed whether the CTLs recognized TAPC expressing the recipient's HLA alleles. We found moderate reactivity of the CTL with 1–5 TAPC expressing recipient HLA alleles. Taken together, our data indicate that reactivity of virus-specific CTLs with hematopoietic APC does not correlate with the risk of developing GvHD, and that virus-specific CTL can safely be infused into HLA class I and/or II mismatched recipients. Disclosures:No relevant conflicts of interest to declare.

Targeting Multiple Myeloma with Human Ig Light Chain Specific Cytotoxic T Lymphocytes

AbstractAbstract 3018The variable regions of Ig expressed by malignant B cells can serve as a tumor specific antigen. Clinical trials of idiotype (Id) vaccines have demonstrated humoral responses and prolonged remission duration in a recent phase III trial of follicular lymphoma patients in the first remission (Schuster et al, J Clin Oncol 27: 793S, 2009). However, the potentially immunogenic epitopes derived from Ig that stimulate CD8+ T cell immunity have been incompletely characterized. Here, we identified nine out of 14 candidate peptides derived from the Ig L-chain variable region of the human U266 myeloma line, which generated cytotoxic T lymphocytes (CTLs) from 53 HLA A2+ normal donors. These CTLs lines, as well as CTLs line isolated from myeloma patients by stimulation with autologous L-chain Id peptides, specifically produced IFN-γ in response to peptide-pulsed T2 cells and lysed U266 and autologous myeloma cell targets, respectively, but not normal blood B cells. Lysis was HLA class I-dependent, suggesting that primary myeloma cells express Id peptides on the cell surface in combination with HLA molecules. Nine CD8+ Id peptide-specific T-cell clones exhibited the effector memory phenotype and the ability of these T cell clones to eliminate U266 tumor in immune deficient mice is being tested. Finally, sequence analysis revealed shared T-cell epitopes in both framework and CDR regions of the U266 L-chain. CTLs generated against a shared U266 epitope lysed patient-derived myeloma cells expressing the shared sequence, suggesting a strategy to overcome the limitation of patient-specific Id vaccine manufacture. Our data identified novel immunogenic Id L-chain T-cell determinants and suggests that, unlike previously described Ig heavy chains, these sequences harbor common T-cell epitopes that may provide the rationale for shared Id vaccines. Disclosures:No relevant conflicts of interest to declare.

Administration of Tumor-Specific Cytotoxic T Lymphocytes Engineered to Resist TGF-ß to Patients with EBV-Associated Lymphomas

AbstractAbstract 560EBV-associated Hodgkin's Lymphoma (HL) and some non-Hodgkin's lymphomas (NHL) express EBV proteins, which are potential targets for immunotherapy. Lymphomas arising in the immune-competent host, however, utilize multiple immune evasion strategies to evade host T cell responses. These include limited expression of EBV antigens, so that only the subimmunodominant EBNA1 and LMP1 and 2 antigens are expressed (type II latency); and production of immunosuppressive cytokines, such as TGF-ß. We first tested our hypothesis that cytotoxic T lymphocytes (CTL) enriched for specificity to LMP antigens would have efficacy in patients with EBV+ lymphoma. 36 patients with EBV+ HL and NHL received LMP CTLs generated using antigen-presenting cells genetically modified to overexpress either LMP2 alone (n=16), or LMP2 and inactive LMP1 (ΔLMP1) (n=20). No immediate toxicity was observed following infusion. Of the evaluable patients who have completed 8 weeks of follow-up, 17 were high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment. 16 of 17 remain in remission for a median of 2.5 years (range 6 months to >5 years). Another 16 patients had detectable disease at the time of CTL infusion, and 11 had clinical responses (78%), including 8 (57%) complete responses. The median duration of the clinical responses is 1.5 years. However, 36% of patients with active disease were either resistant to CTL therapy (n=3) or relapsed/progressed >9 months after CTL therapy (n=2), suggesting the importance of additional tumor immune escape mechanisms. TGF-ß inhibits the growth and function of effector T cells and is secreted both by lymphoma cells and by infiltrating T regulatory cells. To test our hypothesis that the infused CTL can be made resistant to the TGF-ß produced by these tumors, LMP-CTL from 7 patients with relapsed EBV+ve HL, were transduced with a retrovirus vector encoding the dominant-negative TGF-ß type II receptor (DNR). Unlike non-transduced CTL, DNR-transduced CTL were resistant to the anti-proliferative effects of recombinant TGF-ß in vitro. These 7 CTL lines were predominantly CD45RAneg/CD62Lneg (mean 43%;range 1–86%) with a mean of 20% (range 1–48%) of T cells showing a CD45RAneg/CD62Lpos phenotype. Transduction efficiency ranged from 11–30% (median 20%). 3 patients have been treated with DNR-transduced LMP-CTL receiving a dose range of 4 to 10 × 107CTLs/m2 without toxicity. A 2–7 fold increase in EBV/LMP2 –specific T cells was seen by 3 months post CTL infusion, measured as increased numbers of T cells responding in IFN-γ Elispot assay. DNR-transduced T-cells have been detected by quantitative real-time PCR amplification in the peripheral blood of all patients for a median of 7 months (range 2 to >12) post infusion. One patient who had an incomplete response to LMP-CTL alone attained a sustained complete remission, one patient had a partial response and one patient a mixed clinical response. Hence, immunotherapy with CTL targeting LMP antigens is well tolerated in patients with EBV+ lymphoma and can induce durable clinical responses. Furthermore, for patients resistant or with incomplete responses to CTL therapy, the use of DNR-transduced CTL may be beneficial. Disclosures:No relevant conflicts of interest to declare.

A CD8+ T-Cell Clone Directed Against AML Blasts that Recognizes a Tumor Specific Antigen Expressed Also by Solid Tumors

AbstractAbstract 4289The aim of this study was to generate cytotoxic T-lymphocytes (CTL) clones directed against AML cells and to identify new immunogenic antigens with the following properties: i) to be overexpressed by leukemic cells and not by normal tissues; ii) to be shared among different AML subtypes; iii) to play a role in leukemic growth/survival; iv) to be expressed by leukemic stem cells.To this end, we loaded normal dendritic cells (DC) from a healthy donor with apoptotic bodies from primary AML cells and used loaded DC to stimulate autologous lymphocytes (i.e. lymphocytes from the donor). Donor was selected to be partially matched with the leukemic patient for MHC-class I, in that he shared two MHC-class I alleles at the HLA-supertype level (HLA-B7 and HLA-B44). With this strategy, a CD8+ T-cell line was generated that recognized both loaded DC and AML cells used for loading, but not DC loaded with normal cells derived from the patient (PHA-blasts). This CTL line was cloned by limiting dilutions and 180 clones were screened by IFN-g elispot against 2 different AML samples that were expressing both HLA-B7 and HLA-B44; one additional mismatched AML sample was used as negative control. Two clones were selected that recognized HLA-B7+/HLA-B44+ AML cells but not the negative control (namely, clone 31D3 and 8E12). To determine the HLA-restriction element and to verify shared antigen expression among AML subtypes, we tested both clones against a panel of 18 HLA-typed primary AML samples. We found that clone 31D3 was restricted by HLA-B7 and clone 8E12 by HLA-B44. Each clone recognized 5/5 HLA-matched AML samples of different subtype. In addition, the clones did not recognize both resting and activated normal myeloid, nor lymphoid and CD34+ cells expressing the proper HLA-restriction allele. In addition to elispot activity, both clones showed killing of AML cells in a CFSE-based cytotoxic assay. To confirm the lack of reactivity against normal tissues, we analyzed the activity of both clones against HLA matched or mismatched fibroblasts and we found that clone 8E12 displayed a low reactivity activity against normal matched fibroblasts, while clone 31D3 was not reactive.We then focused the analysis on clone 31D3 and tested whether its activity was restricted to leukemia or also directed against a panel of HLA-B7 positive (N=3) or negative (N=5) solid tumors. Interestingly, we found that the clone 31D3 could recognize one colon carcinoma and one melanoma cell line expressing the HLA-B7 supertype. In particular, the melanoma cell line (G4-mel) was HLA-B35+ (HLA-B35 belongs to the HLA-B7 supertype) and was recognized at very high levels. The availability of a tumor cell line expressing adequate amounts of antigen will make the generation of a cDNA library for antigen identification more feasible than with primary leukemic cells. Finally, to further confirm that HLA-B35 is the proper MHC restriction element and to determine to what extent the antigen is shared among different tumors, we transduced 8 tumor cell lines, 3 normal B-lymphoblastoid and 1 fibroblast cell line with the HLA-B35 allele. Clone 31D3 efficiently recognized 6/8 HLA-B35 transduced tumor cell lines (1 AML, 2 melanomas, 2 colon and 1 breast carcinoma) but none of the control cell lines.In conclusion, clone 31D3 is restricted by the HLA-B7 supertype, it recognizes an antigen that is expressed by leukemic cells and not by normal tissues and, most importantly, is shared among tumors of different histology. Since the vast majority of malignant cells tested (both primary cells and in vitro established cell lines) were targeted by this clone, the putative antigen might be a protein playing an important role in tumor growth or survival. We are now testing the activity of the clone against purified leukemic stem cells and attempting to identify this shared tumor antigen. Disclosures:No relevant conflicts of interest to declare.

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.

Peptides derived from human insulin‐like growth factor‐II mRNA binding protein 3 can induce human leukocyte antigen‐A2‐restricted cytotoxic T lymphocytes reactive to cancer cells

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.

Lovastatin Inhibits T-cell Proliferation While Preserving the Cytolytic Function of EBV, CMV, and MART-1-specific CTLs

Statin treatment has been shown to reduce graft-versus-host disease while preserving graft-versus-tumor effect in allogeneic stem cell transplantation. Herein, we investigated whether lovastatin treatment affects the function of human cytolytic T lymphocytes (CTLs). Upon T-cell receptor stimulation, lovastatin significantly inhibited the proliferation of both CD4+ and CD8+ T cells from healthy donors whereas their intracellular cytokine production including interferon-γ and tumor necrosis factor-α remained the same with a slight decrease of interleukin-2. Moreover, the specific lysis of target cells by CTL lines derived from patients and normal donors specific for Epstein-Barr virus-encoded antigen latent membrane protein-2 or cytomegalovirus-encoded antigen pp65 was uncompromised in the presence of lovastatin. In addition, we evaluated the effect of lovastatin on the proliferation and effector function of the CD8+ tumor-infiltrating lymphocytes (TILs) derived from melanoma patients specific for MART-1 antigen. Lovastatin significantly reduced the expansion of antigen-specific TILs upon MART-1 stimulation. However, the effector function of TILs, including the specific lysis of target cells and secretion of cytokine interferon-γ, remained intact with lovastatin treatment. Taken together, these data demonstrated that lovastatin inhibits the proliferation of Epstein-Barr virus, cytomegalovirus, and MART-1-specific CTLs without affecting cytolytic capacity. The differential effect of lovastatin on the proliferation versus cytotoxicity of CTLs might shed some light on elucidating the possible mechanisms of graft-versus-host disease and graft-versus-tumor effect elicited by alloimmune responses.

A new bioassay for the immunocytokine L19-IL2 for simultaneous analysis of both functional moieties

Currently, cancer directed new biological entities (NBEs) in the pharmaceutical R&D pipelines are derived from monoclonal antibodies in various formats, such as immunocytokines. Generally, immunocytokines are bi-functional molecules that consist of a specific targeting antibody-based portion and a linked cytokine. To confirm the quality of the drug product both moieties have to be characterized using appropriate techniques. Until now, the binding capacity of antibodies is usually examined by ligand binding assays whereas the biological activity of the linked cytokine is determined by cell-based potency assays. However, the simultaneous analysis of both functional moieties in a single assay format has not been described so far. In this paper we present a newly designed bioassay format for the anti-cancer immunocytokine L19-IL2, comprising of the human vascular targeting single-chain Fv L19 and human interleukin 2 (IL2). This new potency assay allows simultaneous analysis of both moieties, thus specific L19 binding capacity and the ability of IL2 to induce the proliferation of the detector cytotoxic T-cell line CTLL-2. Assay development was performed with special focus on application of different fitting models for the sigmoid dose–response curves to evaluate the influence of model optimization on the validity of assay results. For assay validation generally accepted characteristics were determined. Assay specificity was shown by testing L19-IL2 related compounds. All other validation parameters were derived from 25 batch runs using five nominal L19-IL2 concentrations, covering a range from 60% to 140% of the standard's potency. Accuracy ranged from −3.4% to −6.9% relative error (%RE). Interbatch precision ranged from 6.1% to 10.6% coefficient of variation (%CV). For assay linearity a coefficient of determination ( R 2) of 0.9992 was found. Assay robustness was shown with L19-IL2 samples after three freeze–thaw cycles and also with different cell passages of the used cytotoxic T-cell line. Based on the data, we conclude that this assay is valid for potency estimation of the immunocytokine L19-IL2. Moreover, this format represents a major improvement compared to other approaches which only allow potency evaluation of both functional moieties in separate assays. In general the underlying assay principle described seems suitable for potency determination of other immunocytokines.

Allogeneic T-Cell Therapy for Epstein-Barr Virus-Positive Posttransplant Lymphoproliferative Disease: Long-Term Follow-Up

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) is an important cause of morbidity and mortality in transplant patients. EBV-specific cytotoxic T-cell line (CTL) that are in vitro grown and donor derived have been used to prevent and treat EBV-positive PTLD in hematopoietic stem-cell transplant recipients (1). However, donor-derived T cells are neither generally available nor appropriate for PTLD arising after solid organ transplantation. Furthermore, although monitoring EBV load in blood may indicate a risk of PTLD after hematopoietic stem-cell transplant, allowing time for CTL preparation, this monitoring gives less convincing results after solid organ transplantation. Additionally, the alternative strategy of generating autologous CTL after PTLD diagnosis often produces unacceptable delays in treatment. To counter these problems, we generated a bank of 100 EBV-specific CTL from healthy blood donors covering more than 95% of common UK human leukocyte antigen (HLA) haplotypes and treated a patient with PTLD with a partially HLA-matched CTL line that resulted in complete tumor regression (2, 3). Subsequently, we used these cell lines in a phase 2 multicenter clinical trial to treat PTLD in a best HLA match basis (4). All 33 trial participants had progressive disease, despite conventional treatments. Our trial recorded a response rate of 52% at 6 months, with 14 patients achieving complete remission (CR), three patients with partial remission (PR), and 16 patients showed no response (NR); five of whom died during the CTL treatment. Significantly better response rates were seen in patients with closer HLA matches and higher numbers of CD4+ T cells in the infused CTL. We now report the long-term outcome of trial participants. We obtained follow-up data from 32 of the 33 trial participants, 4 to 9 years after their last CTL infusion. At 6 months, 15 of the 32 patients showed NR and 17 patients were responders (three PR and 14 CR). All those in the PR and NR groups, but none of the CR group, received further PTLD treatment after CTL therapy. Nineteen (59%) of the 32 patients are alive to date and 13 (41%) have died. Of the 14 patients who achieved CR at 6 months, 12 patients (86%) survived and are still in CR after 4 to 9 years. Two patients (14%) from the CR group had died; one at 10 months post-CTL therapy from relapsed PTLD and the other at 5 years from a chest infection while PTLD was in CR. Of the 19 surviving trial participants, 13 participants (68%) were responders (one PR and 12 CR) and six participants (32%) were NR at 6 months. In contrast, of the 13 patients who have died, nine patients (69%) were NR and four patients (31%) were responders (two PR and two CR) at 6 months. There was a significantly increased survival rate among the PR and CR group compared with the NR group (P=0.018; Fig. 1).FIGURE 1.: The Kaplan-Meier overall survival rate. There was a significantly increased survival rate in patients who achieved complete response (CR) and partial response (PR) compared with those who showed no response (NR) to cytotoxic T-cell (CTL) therapy (Log-rank test; P=0.018).Our clinical trial showed allogeneic T-cell therapy for PTLD to be safe and effective in the short term, and this 4- to 9-year follow-up data are important in showing that, although of indeterminate life-span in vivo, CTL induces long-term remission of PTLD in patients with refractory disease. Therefore, it seems appropriate to use this therapy early in PTLD and for prophylaxis in high-risk cases. We are generating a new bank of clinical-grade EBV-specific CTL under good manufacturing practice conditions to provide partially matched CTL internationally on a not-for-profit basis, funded by The Wellcome Trust, UK. Tanzina Haque1 Karen A. McAulay2 Deirdre Kelly3 Dorothy H. Crawford2 1 Department of Virology Royal Free Hospital UCL Medical School London, United Kingdom 2 Clinical and Molecular Virology Laboratory University of Edinburgh Edinburgh, United Kingdom 3 Liver Unit Birmingham Children's Hospital Birmingham, United Kingdom