ALBERT

Description: Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3′-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3′-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3′-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host.

Description: Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection. These obligatory structures exposed to the host immune system provide important targets for rational vaccine design. Here, we use a systematically designed series of multi-domain constructs in combination with small angle X-ray scattering (SAXS) to determine the structure of the main immunoreactive region from a major antigen from Leptospira interrogans, LigB. An anti-LigB monoclonal antibody library exhibits cell binding and bactericidal activity with extensive domain coverage complementing the elongated architecture observed in the SAXS structure. Combining antigenic motifs in a single-domain chimeric immunoglobulin-like fold generated a vaccine that greatly enhances leptospiral protection over vaccination with single parent domains. Our study demonstrates how understanding an antigen’s structure and antibody accessible surfaces can guide the design and engineering of improved recombinant antigen-based vaccines.

Description: Abstract 4476 Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HSCT). It is not known why some patients develop cGVHD and others do not, but identifying predictive biomarkers would facilitate the development of pre-emptive therapeutic strategies. Investigation into the pathophysiology of cGVHD has revealed different patterns of immune reconstitution in patients who develop cGVHD following HSCT. This suggests that the development of cGVHD occurs as a result of altered immune homeostasis and the inability to establish B and T cell tolerance. We prospectively examined 88 patients with hematologic malignancies following allogeneic HSCT at the Brigham and Woman's Hospital/Dana-Farber Cancer Institute between the years 2004 and 2008 and measured plasma cytokines known to modulate effector T cell, regulatory T cell, and B cell homeostasis and function. Seventy-six patients (86%) received reduced intensity conditioning; 84 (95%) received filgrastim-mobilized peripheral blood stem cells. Median follow-up for all patients was 5 years (range 2.9 to 7.3 years). Fifty-nine percent developed cGVHD. Plasma samples were collected at 1, 3, 6, and 12 months after HSCT and multiplex Luminex bead assays were used to measure levels of the following cytokines: Interferon-γ, IL-2, IL-7, IL-1β, IL-12, TNFα, IL-4, IL-5, IL-6, IL-10, and GM-CSF. Cytokine levels in patients who developed cGVHD were compared to patients who did not develop cGVHD. Results are shown in figure 1. Significantly higher levels of IL-4, IL-6, IL-12, and IL-1β were observed at 1 and/or 3 months after transplantation in patients who subsequently developed cGVHD. IL-4 and IL-6 are characteristic of T-helper-2 (TH2) cellular and humoral immune responses previously associated with cGVHD and fibrogenesis. IL-12 and IL-1β have heterogeneous functions including stimulating interferon-γ and TNFα production, enhancing cytotoxic and helper T cells, and promotion of autoimmunity. IL-1α also stimulates IL-6 production and activates fibroblasts. Finally, GM-CSF was significantly elevated in patients who do not develop cGVHD at 1 year following HSCT (2.5 v 1.46 pg/mL, p=0.017). As shown in figure 1, we observed a general pattern for many cytokines, including IFNγ, IL-2, IL-4, IL-5, IL-6, and IL-1β, in which these cytokines are initially similar in both groups, or lower in patients without cGVHD, but gradually increase over time in patients who do not develop cGVHD. In contrast, cytokine levels remain stable in patients with cGVHD and are generally lower 1 year after transplantation in these patients. These late differences may reflect immune suppressive therapies as well as persistent abnormalities of immune homeostasis. Taken together, these results support the hypothesis that alterations in immune homeostasis early after allogeneic HSCT are significantly associated with the subsequent development of cGVHD. Manipulation of the cytokine environment early after HSCT can modulate immune homeostasis and may be of potential prophylactic value. Administration of homeostatic cytokines late after HSCT may also have therapeutic benefit. Disclosures: No relevant conflicts of interest to declare.

Description: Acute GVHD (aGVHD) remains a major source of morbidity after allogeneic hematopoietic cell transplantation. CD30 is a cell-surface protein expressed on certain activated T cells. We analyzed CD30 expression on peripheral blood T-cell subsets and soluble CD30 levels in 26 patients at the time of presentation of aGVHD, before the initiation of treatment, compared with 27 patients after hematopoietic cell transplantation without aGVHD (NONE). Analysis by flow cytometry showed that patients with aGVHD had a greater percentage of CD30 expressing CD8+ T cells with the difference especially pronounced in the central memory subset (CD8+CD45RO+CD62L+): GVHD median 12.4% (range, 0.8%-33.4%) versus NONE 2.1% (0.7%, 17.5%), P 〈 .001. There were similar levels of CD30 expression in naive T cells, CD4+ T cells, and regulatory (CD4+CD127lowCD25+) T cells. Plasma levels of soluble CD30 were significantly greater in patients with GVHD: median 61.7 ng/mL (range, 9.8-357.1 ng/mL) versus 17.4 (range, 3.7-142.4 ng/mL) in NONE (P 〈 .001). Immunohistochemical analysis of affected intestinal tissue showed many CD30+ infiltrating lymphocytes present. These results suggest that CD30 expression on CD8+ T-cell subsets or plasma levels of soluble CD30 may be a potential biomarker for aGVHD. CD30 may also represent a target for novel therapeutic approaches for aGVHD.

Description: Umbilical cord blood (UCB) is an alternative source of hematopoietic stem cells (HSC) for allogeneic HSC transplantation but reconstitution of T cell immunity remains problematic even with double UCB transplantation (dUCBT). This is related to the small T cell numbers of the UCB graft, the naïve nature of these T cells and often the use of ATG as part of the conditioning regimen. Consequently, UCBT recipients are susceptible to viral infections that require intact T cell immunity for their containment. Epstein Barr virus (EBV) is a gammaherpes virus that is transmitted orally. EBV initially replicates in a lytic cycle in the epithelium of the oropharynx and then establishes a latent state in memory B cells. Reactivation of EBV from latent to lytic cycle is mediated by physiologic stress of the memory B cell pool and by defects in antiviral T cell immunity, and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. The specific immunological correlates of EBV reactivation in adult UCBT recipients have not been examined. In the present study we attempted to distinguish immune reconstitution profiles in UCBT recipients who developed EBV viremia from those who did not, in order to better understand the immune profile of UCBT recipients who control gammaherpes virus reactivation. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. EBV viral load was determined on a weekly basis after dUCBT. Immunophenotype of peripheral blood lymphocytes, serum cytokine levels and T cell receptor excision circle analysis (TREC) values, were examined prior to and at 1, 2, 3, 6 and 12 months after UCBT and were compared between EBV viremic and non-viremic patients using the Wilcoxon-rank sum test. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients who developed EBV viremia displayed higher numbers of CD19+ B cell numbers (p=0.04) and CD4+CD25+ T regulatory cells (p=0.03) compared with patients who never became viremic. Surprisingly, development of EBV viremia correlated with increased numbers of CD3+ (p=0.04), CD4+ (p=0.015) and CD8+ (p=0.021) T cells. This finding was counterintuitive, as one might expect better quantitative peripheral T cell reconstitution in patients with recovering immunity and no EBV reactivation. One mechanism of impaired immune reconstitution after UCBT is skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined whether development of EBV viremia was associated with altered naïve versus memory T cell distribution. We determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p=0.0023; CD8+CD45RO+, p=0.019) at two months after dUCBT. Neither development nor control of EBV viremia correlated with TREC recovery, which occurred after the time period of EBV viremia. We were unable to identify EBV-specific T cells in any patient group due to the very low T cell numbers during the time of viremia. However, analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p=0.03) and lower clonality (p=0.03), among patients who did not develop EBV reactivation, compared with those who developed EBV viremia and PTLD. Assessment of serum levels of SCF (a c-kit ligand), IL-7, thrombomodulin, VEGF and angiopoetin-1 showed that patients without EBV viremia had significantly higher levels of SCF (p=0.0001) and IL-7 (p=0.05), at 1 and 2 months after dUCBT, compared with patients who developed EBV reactivation and PTLD. SCF-mediated signaling via c-kit is integral to the homing and longevity of uncommitted hematopoietic stem cells whereas IL-7 is a critical factor for survival and homeostasis of naïve T cells. Together our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naïve T cell homeostasis, which enables maintenance of a diverse TCR repertoire. Disclosures: No relevant conflicts of interest to declare.

Description: The treatment of hematological malignancies with umbilical cord blood transplantation (UCBT) is rapidly increasing for adult patients. Disadvantages of UCBT include insufficient cell numbers for adult patient reconstitution, a lack of antigen experienced cells, and deficits in T cell signal transduction mechanisms. Consequently, UCBT is frequently associated with impaired immune function and high infection-related mortality. To counter these difficulties, transplantation with two UCB units has been employed to improve immune reconstitution in adult patients. We evaluated both the quantitative and functional reconstitution of cellular immunity in a group of adult patients undergoing UCBT. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. The grafts were at least a 4/6 match with each other and the recipient. Here we report the results of 27 patients who have completed at least one year of follow up. Assessments were done prior to transplant and at various time intervals until 12 months post UCBT. Neutrophil and platelet engraftment occurred at a median of 21 days and 42 days, respectively. CD3+ populations remained severely depressed until 8 wks post-transplant when they gradually began to re-emerge. However the CD4+ and CD8+ populations demonstrated distinctly different reconstitution kinetics. At 6 months the median value of absolute numbers of CD4+ lymphocytes was 35% of pre-transplant levels increasing to 42% at 1 yr post-transplant, a median value far below the normal range for adults. In contrast, at 6 months post-transplant CD8+ lymphocytes remained severely depressed to 12% of pre-transplant levels, but dramatically increased and reached normal levels by 1 yr after UCBT. Interestingly, both the CD14+ monocyte and the CD16+CD56+ NK cell populations expanded dramatically at 4 wks post-transplant and reached pre-transplant levels and were within the normal range by 6 months. CD20+ B cell repopulation began at 8 wks post-transplant, displayed a striking expansion leading to a 17-fold increase in the median value for B cell numbers over pre-transplant values at 1 year and resulting in a median value of absolute numbers near the top of the normal range. To evaluate functional T cell immune reconstitution in vivo, we performed IFN-γ ELISpot analysis on CMV stimulated PBLs and compared the results to a PCR-based assay for CMV viremia. Additionally, we assessed the reconstitution of thymopoiesis with the T cell receptor excision circle (TREC) assay and real-time PCR. 16/27 patients and 26/52 UCB products were CMV seropositive prior to transplant. In the post-UCB period, development of CMV-specific effectors as determined by ELISpot did not always correlate with clearance of CMV viremia. Specifically, prior to 8 weeks post-UCBT, 8 out of 12 (67%) patients with CMV-positive ELISpot displayed CMV viremia, between 8 weeks and 100 days post-UCBT 4 out of 11 (36%) patients with CMV-positive ELISpot displayed CMV viremia and after 100 days post-UCBT only 3 out of 10 patients (30%) who developed CMV-positive ELISpot remained positive for CMV viremia. Identification of functional CMV effectors was only associated with the numbers of CD8+CD45RA+ cells (p=0.01) and the development of high TREC concentrations (p=0.01) that were detected after 6 months of UCBT, and was independent of GvHD or mixed chimerism. Taken together these results indicate that reconstitution of T cell immunity after UCBT is characterized by delayed recovery of CD4+ and CD8+ T cells and correlates with reconstitution of thymopoiesis and increase of naïve CD8+CD45RA+ T cells that can develop into efficient pathogen-specific effectors.

Description: Umbilical cord blood grafts are increasingly used as sources of hematopoietic stem cells in adults. Data regarding the outcome of this approach in adults are consistent with delayed and insufficient immune reconstitution resulting in high infection-related morbidity and mortality. Using cytomegalovirus (CMV)–specific immunity as a paradigm, we evaluated the status, mechanism, and clinical implications of immune reconstitution in adults with hematologic malignancies undergoing unrelated double unit cord blood transplantation. Our data indicate that CD8+ T cells capable of secreting interferon-γ (IFN-γ) in a CMV-specific enzyme-linked immunosorbent spot (ELISpot) assay are detectable at 8 weeks after transplantation, before reconstitution of thymopoiesis, but fail to clear CMV viremia. Clearance of CMV viremia occurs later and depends on the recovery of CD4+CD45RA+ T cells, reconstitution of thymopoiesis, and attainment of T-cell receptor rearrangement excision circle (TREC) levels of 2000 or more copies/μg DNA. In addition, overall survival was significantly higher in patients who displayed thymic regeneration and attainment of TREC levels of 2000 or more copies/μg DNA (P = .005). These results indicate that reconstitution of thymopoiesis is critical for long-term clinical outcome in adult recipients of umbilical cord blood transplant. The trial was prospectively registered at http://www.clinicaltrials.gov (NCT00133367).

Description: CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the control of chronic graft-versus-host disease (cGVHD). In this study, we examined telomere length and telomerase activity of Treg and conventional CD4+ T cells (Tcon) in 61 patients who survived more than 2 years after allogeneic hematopoietic stem cell transplantation. Cell proliferation and expression of Bcl-2 were also measured in each subset. Treg telomere length was shorter and Treg telomerase activity was increased compared with Tcon (P 〈 .0001). After transplantation, Treg were also more highly proliferative than Tcon (P 〈 .0001). Treg number, telomerase activity, and expression of Bcl-2 were each inversely associated with severity of cGVHD. These data indicate that activation of telomerase is not sufficient to prevent telomere shortening in highly proliferative Treg. However, telomerase activation is associated with increased Bcl-2 expression and higher Treg numbers in patients with no or mild cGVHD. In contrast, patients with moderate or severe cGVHD have fewer Treg with lower levels of telomerase activity and Bcl-2 expression. These results suggest that failure to activate Treg telomerase may restrict proliferative capacity and increase apoptotic susceptibility, resulting in the loss of peripheral tolerance and the development of cGVHD.

Description: Abstract 3316 Poster Board III-204 Background Considerable evidence suggests that immune responses after allogeneic hematopoietic stem cell transplantation (HSCT) play a major role in the prevention of disease relapse, called the graft-versus-tumor (GVT) effect. In patients who relapse after reduced-intensity conditioning (RIC) HSCT, donor lymphocyte infusion (DLI) may boost donor-T cell chimerism and induce a therapeutic GVT effect. Since the advent of imatinib mesylate for chronic myeloid leukemia (CML), DLI is now mostly given to patients with hematological diseases other than CML known to be less responsive to adoptive immunotherapy. Objective Our intent was to study the effect of infused T cell subsets on immune reconstitution and clinical outcomes such as acute and chronic graft versus host disease (aGvHD and cGvHD, respectively), overall survival (OS), and progression free survival (PFS) in patients with relapsed hematological malignancies other than CML who underwent RIC HSCT. Methods The DLI products given to 22 patients with relapsed/persistent hematological diseases (8 Hodgkin lymphoma, 4 myelodysplastic syndrome, 2 acute myeloid leukemia, 2 chronic lymphocytic leukemia, 2 Non-Hodgkin lymphoma, 1 acute lymphoblastic leukemia, 1 plasma cell myeloma, 2 polycythemia vera) who received matched related or unrelated DLI after HSCT were analyzed for CD3+, CD4+, and CD8+ T cell subsets. Patients were treated with a consistent RIC with fludarabine (120mg/m2 total) and intravenous busulfan (3.2mg/kg total) at our institution from 2005 to 2008. GVHD prophylaxis in the majority of patients (77.3%) consisted of tacrolimus, sirolimus and methotrexate. Peripheral blood (PB) obtained before and after DLI was analyzed for CD3+ CD4+, CD8+, CD19+, CD20+, and CD14+ cells. The lymphocyte subset contents of DLI and changes of patients' immune reconstitution before and after DLI were correlated with clinical outcomes such as aGVHD, cGVHD, OS, and PFS. Results The 2-year OS and PFS after DLI were 41% and 22%, respectively, with a median follow-up of 1.1 years for survivors. The 2-year-relapse rate was 77.8%, but treatment-related mortality (TRM) was 0%. A complete response was achieved in 18% of the patients. The cumulative incidences of grade II-IV aGVHD and cGvHD were 9.1% at 200 days and 27.8% at 2 years after DLI, respectively. A significant decrease in CD4+ (p=0.01) and a significant increase in PB CD8+ (p=0.04) cell frequency were observed after DLI infusion. Infused total and lymphocyte subset cell dose had no effect on any of the clinical outcomes. Higher pre-infusion PB CD4+ (p=0.04, HR 0.89) and lower pre-infusion CD14+ cell (p=0.03, HR 1.09) frequencies in the recipient were associated with better OS. Lower pre-infusion CD14+ cell frequency was associated with better PFS (p=0.003, HR 1.19). In addition, increased pre-to-post DLI CD19 and CD20 frequencies were associated with PFS (p=0.03, HR=1.21; p=0.04, HR=1.16, respectively). None of the changes in immune parameters after DLI were associated with cGVHD. Conclusion Clinical responses to DLI in patients with relapsed hematological malignancies other than CML remain unsatisfactory, although the associated risk of GVHD and TRM are low. The dose of CD3, CD4 and CD8 cells in the DLI product was not correlated with clinical outcome. However, pre-infusion immune status and demonstration of significant changes in immune reconstitution after DLI can affect clinical outcomes. Strategies to modulate pre- and post-immune status of the recipients warrant further investigation to improve the efficacy of DLI. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3426 Poster Board III-314 Lenalidomide is an immunomodulatory drug recently reported to have an objective response rate (ORR) of 35-53% in relapsed/refractory CLL, even with poor risk features. Initial therapy of CLL currently includes chemoimmunotherapy combinations like FCR, which have high response rates and improved PFS, but at the cost of myelosuppression and infection. Given the high ORR reported with lenalidomide and its potential to spare immune function, we undertook this Phase I study to explore the safety and tolerability of lenalidomide in combination with fludarabine and rituximab. Eligibility criteria included a confirmed diagnosis of CLL/SLL, previously untreated with systemic therapy with an indication for therapy by NCI-WG 1996 criteria; ANC 〉 1000, platelets 〉 50K, and adequate organ function. Six dose levels were planned, beginning with fludarabine 25 mg/m2 days 1-3, rituximab 375 mg/m2 day 1, and lenalidomide 2.5 mg administered on days 1-21 of a 28-day cycle. The study used a standard 3+3 dose escalation design, with dose limiting toxicity (DLT) assessed in the first 28 days and defined as grade 3 or greater non-hematologic toxicity, grade 4 neutropenia or thrombocytopenia, grade 3 febrile neutropenia, or a 〉2-week delay in initiating the second cycle. Nine patients were enrolled on this study, 7 males and 2 females, with a median age of 59 yrs (range 37-66). The median time from diagnosis to study entry was 66.1 months (range 11.7-82.8 months). The median WBC at study entry was 99.6 (range 11.1-325.7). Six patients had Rai 3-4 disease, and three patients had bulky lymphadenopathy (〉5 cm) on physical exam. Median beta-2 microglobulin level at study entry was 3.8 mg/L (range 2.5-7.7). The first cohort enrolled four patients, of whom two developed DLTs (grade 4 neutropenia persisting through day 50; febrile syndrome with grade 3 rash, myalgias and grade 4 CPK elevation). The study then proceeded to enroll five patients at dose level -1, with the same dosing of rituximab and fludarabine, but reduced lenalidomide dosing of 2.5 mg every other day for 21 of 28 days. Only two of five patients on dose level -1 completed the planned six cycles of therapy. The other three patients discontinued after 3 cycles due to toxicity: persistent grade 2 thrombocytopenia preventing further therapy; recurrent grade 3-4 neutropenia and thrombocytopenia, despite growth factor support, dose reduction and holding lenalidomide; and intermittent recurrent grade 3 tumor flare, rash and hand-foot syndrome, along with recurrent grade 3 neutropenia despite similar measures as above. At that point the study was closed to enrollment due to the significant myelotoxicity and idiosyncratic tumor flare, resulting in only two of nine patients completing the planned therapy. The median number of cycles completed for all patients was three. Six of the nine enrolled patients were evaluable for response, with five of nine having objective responses (56%, 90% CI 25-83%). No significant difference was observed in IgG and IgM levels between baseline and two months on study, although a trend toward a decrease in IgA levels (p=0.05) was observed. Baseline CD4 counts were variable (median 1139, range 529-2687), but showed significant declines of 574 cells by week 4 (p=0.003) and 707 cells by post-treatment (p=0.002). Analysis of alterations in serum cytokines is ongoing. We conclude that administering lenalidomide concurrently with fludarabine and rituximab is difficult, does not appear to preserve immune function, and limits the ability to deliver adequate therapy with either drug. Other trials currently in progress are exploring alternative schedules of lenalidomide administration with fludarabine and other standard CLL chemotherapy regimens; our data would favor a sequential schedule. Disclosures Brown: Celgene: Honoraria, Provided funding for this investigator-initiated study; Genzyme: Research Funding; Genentech: Consultancy; Calistoga: Consultancy. Off Label Use: Lenalidomide for CLL. Hochberg:Enzon: Speakers Bureau; Biogen-Idec: Speakers Bureau; Genentech: Speakers Bureau; Amgen: Speakers Bureau.