ALBERT

Description: Background: Patients with myelodysplastic syndromes (MDS) display several abnormalities of T cell and NK cellular immunity. Tumor lysis by NK cells occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs. Lenalidomide is an immunomodulatory (IMiD) drug structurally related to thalidomide, which has proven clinical efficacy for the treatment of low-risk MDS (List et al, NEJM2005; 352:549). Investigations in patients with multiple myeloma (MM) reported that thalidomide was associated with enhanced cytolytic function and increased NK cells in responding patients. We investigated the action of lenalidomide on NK function and activating NK receptor expression in patients with MDS. Methods: MDS patients were categorized according to WHO category, age, sex, IPSS, and cytogenetics. Peripheral blood cells (PBMCs) were isolated from patients and normal donors and NK receptor expression determined in paired healthy and patient PBMCs by 3-color flow cytometry. NK receptor expression was analyzed for CD158a (KIR2DL1, KIR2DS1), CD158b (KIR2DL2, KIR2DL3, KIR2DL3), KIR3DL1, KIR2DS4, NKG2A, NKG2D, NKp30, NKp44, and NKp46. Cytotoxicity assays were performed using 5-hour 51Cr-release assays. Results: Forty-eight MDS patients and 37 normal donors were analyzed, demonstrating that NK cytolytic function was reduced in the patient population (19% ± 21 S.D. vs. 44% ± 21) (p

Description: Clonal T-cell expansion in patients with T-large-granular lymphocyte (LGL) leukemia occurs by an undefined mechanism that may be related to Fas apoptosis resistance. Here, we demonstrate polarized expansion of CD8+ terminal-memory differentiation in such patients, as demonstrated by CD45RA expression and absence of CD62L expression, suggesting repeated stimulation by antigen in vivo. Elimination of antigen-stimulated T cells normally occurs through Fas-mediated apoptosis. We show that cells from LGL leukemia patients express increased levels of c-FLIP and display resistance to Fas-mediated apoptosis and abridged recruitment of proteins that comprise the death-inducing signaling complex (DISC), including the Fas-associated protein with death-domain (FADD) and caspase-8. Exposure to interleukin-2 (IL-2) for only 24 hours sensitized leukemic LGL to Fas-mediated apoptosis with enhanced formation of the DISC, and increased caspase-8 and caspase-3 activities. We observed dysregulation of c-FLIP by IL-2 in leukemic LGL, suggesting a role in Fas resistance. Our results demonstrate that expanded T cells in patients with LGL leukemia display both functional and phenotypic characteristics of prior antigen activation in vivo and display reduced capacity for Fas-mediated DISC formation.

Description: Abstract 3193 Poster Board III-130 Paroxysmal nocturnal hemogloginuria (PNH) is a clonal genetic disorder associated with multiple mutations in the X-linked phosphatidylinositol glycan class A (PIG-A) gene that causes glycosyl phosphatidylinositol (GPI) anchor protein (AP) deficiency in patients with PNH. Conditional PIG-A gene inactivation (PIG-A (-/-)) in hematopoietic cells of mice does not induce overt proliferative advantage to the HPC compartment. In humans, the molecular basis of clonal expansion of GPI-AP deficient cells is unknown but may relate to selective pressure from the bone marrow microenvironment conducive to GPI-AP survival. Alternatively, coexistence of additional genetic anomalies that confer growth potential has been speculated. A single mutation in the Janus Kinase (JAK)-2 gene has been reported to be the underlying molecular mechanism for myeloproliferative diseases including polycythemia vera (PV), essential thrombocythemia, and myelofibrosis. Substitution of a valine for phenylalanine destabilizes the auto-inhibitory activation domain of JAK2 leading to myeloproliferation. Genetic evidence and in vitro functional studies indicate that V617F gives hematopoietic precursors proliferative and survival advantages. A high proportion of patients with myeloproliferative disorders and myelodysplastic syndrome (MDS) carry this dominant gain-of-function mutation of JAKV617F. The clinical syndromes of PNH and JAKV617F mutations often present as overlapping clinical syndromes raising the possibility that acquired JAKV617F mutations confer growth and survival potential to PNH clones in at least a subset of patients. To address this possibility, 21 PNH patients were screened for the presence of JAKV617F mutations. We show here that the features of Budd-Chiari syndrome (BCS) as well as a predisposition to myelodysplastic syndrome can be caused by an acquired co-existing PIGA and JAKV617F activating mutation. Of the 21 cases examined, PNH with JAK2V617F mutations co-existed in three cases (14.3%). Thus far, two patients have been well characterized. Both cases were male, ages 51 (case 1) and 65 year-old (case 2) with normal cytogenetics and de novo classic PNH manifested by Budd-Chiari syndrome. Flow cytometry assay using CD55, CD59, and CD48 (GPI-APs) and FLAER to detect PNH type cells showed that 90% of granulocytes (case 1) and almost 100% of granulocytes (case 2) were GPI-AP deficient cells. Interestingly, T-cells in both cases displayed a normal phenotype suggesting that the PIGA gene mutation was selectively acquired in a common myeloid progenitor (CMP). From sorted populations of PNH+ and normal type granulocytes (case 1 only) or T cells (case 2), the JAK2V617F mutant clone was found to co-exist with the PIGA mutation using allele-specific polymerase chain reaction (PCR). Real-time quantitative PCR using primers and probe to the junction of exon 5-6 coding region demonstrated drastically low to absent mRNA transcript expression of PIGA only in PNH-type granulocytes in both cases. Individual RT-PCR of the coding region of each exon (2-6) confirmed that transcription was absent in case 1 but limited to regions encoded by exons 2,4, and 6 in case 2. These results suggest that case 2 possesses a splicing mutation involving exon 5 to 6. In conclusion, JAK2V617F mutation is known to cause the constitutive activation of the JAK-STAT signaling pathway, and leads to autonomous cell growth in a cytokine-independent expansion of HPCs. While it has been reported that JAK2V617F mutation occurs in roughly 50% of primary Budd-Chiari syndrome and in 15 patients of 163 (9.2%) (Hoekstra et al, Journal of Hepatology, 6:19, 2009) of patients with PNH, these findings implicate co-existing JAK2V617F /PIG-A mutations may contribute clinically to specific manifestations of splenic venous thrombosis or hepatic vein thrombosis in association with intravascular coagulation and myelodysplasia. Disclosures No relevant conflicts of interest to declare.

Description: Background: The myelodysplastic syndromes (MDS) comprise a spectrum of stem cell malignancies with natural histories that vary from indolent mild cytopenias to rapid transformation to acute leukemia. MDS patients have impaired T cell antigen-induced proliferation and reduced T helper-1 (Th-1) cytokine production. Lenalidomide, an immuno-modulatory drug structurally-related to thalidomide, is FDA-approved for the treatment of MDS with chromosome 5q deletion; however, its mechanism of action is not fully characterized. We hypothesize that immune modulation by lenalidomide will be an effective adjunct to vaccine therapy for patients with MDS. Methods: The immunoregulatory effects of lenalidomide were investigated both in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) from MDS patients and normal controls were stimulated with anti-CD3 cross-linking, allogeneic dendritic cells (allo-DCs), autologous dendritic cells (auto-DCs), and patient-derived autologous bone marrow mononuclear cells (BM-MNC) as antigen sources in the presence of DMSO (vehicle control) and lenalidomide [0.625 μM to 40 μM]. Proliferation of specific CD4+ and CD8+ T cell populations was assessed by Brdu incorporation and intracellular cytokine production by flow cytometry. Preliminary studies were performed to examine the combined effects of the GMCSF/K562 “bystander” vaccine (gift of Dr. I. Borrello, Johns Hopkins University) and lenalidomide on antigen-induced T cell proliferation in PBMC from both normal donors and MDS patients. Results: Lenalidomide augmented a Th-1-biased cytokine (IFN-γ, TNF-α and IL-2) response from normal donors (n=5) and MDS patients (n=5). The Th-1-biased increase in cytokine production accompanied erythroid response in MDS patients treated with 10 mg of lenalidomide for 16 weeks (n=4 responders and 3 non-responders) (List et al, NEJM2005;351:549). Augmentation of antigen-dependent proliferation accompanied cytokine responses both in vitro and in vivo. Next, we examined the effects of lenalidomide on in vitro response to autologous and allogeneic antigens. We found that pre-treatment T cell proliferation in response to auto-DC priming was not distinguishable from background. However, proliferation in response to auto-BM-MNCs used as a source of autologous tumor antigens was significantly increased by lenalidomide in CD3+, CD4+, and CD8+ T cell populations (P=0.002, 0.04, and 0.04, respectively). Proliferation after allo-DC exposure was also significantly enhanced by lenalidomide treatment (P

Description: Abstract 2777 Poster Board II-753 Telomeres are repeated nucleotide sequences at the ends of chromosomes that serve to preserve genetic integrity in hematopoietic progenitors (HPs). Abnormal telomere maintenance resulting from mutations in telomerase or other telomere repair genes have an established pathogenetic role in a subset of acquired and congenital forms of bone marrow failure (BMF). Excess telomere shortening of myeloid elements was reported previously in MDS, however it is unclear whether such abnormalities in telomere maintenance play a pathogenetic role in disease susceptibility or represent a disease specific phenotype arising from accelerated cellular proliferation of the myeloid compartment. Alterations in telomere maintenance and telomerase activity in T cells may offer insight into genetic susceptibility since these cells are not derived from the malignant clone. We examined telomere length and replication history in neutrophils and T cells in newly diagnosed MDS cases (n=66) and compared results to unrelated, age-matched controls (n=63) lacking a prior history of cancer. Telomere length in peripheral blood mononuclear cells was assessed by quantitative real-time PCR (qRT-PCR) and found to be significantly shorter among MDS cases compared to controls. Peripheral blood concentration of the senescence protein stathmin was analyzed in a subset of 48 patients and 48 controls and found to be higher in MDS cases compared to controls after adjustment for age and sex (mean 4.24 pg/ml ± 7.25 in cases vs mean 2.45 pg/ml ± 2.84 in controls, p=0.06) with no change observed with age. Mechanisms of cellular senescence and telomere attrition in T-cells may be related to antigen-driven or homeostatic proliferation. Therefore, telomere length was compared to an independent genetic marker of proliferation, i.e., the concentration of T cell receptor excision circles (TRECs). TRECs are episomal DNA fragments excised during TCR gene rearrangement within the thymus that do not transfer to daughter cells. Therefore, TREC DNA copy number has been shown to be diluted during each round of division and tends to be reduced with age due to impaired generation of new thymic T-cell emigrants. In controls, TREC concentration declined with age consistent with cellular proliferation and loss of thymic function. For every year increase in age, log TREC values decreased by 0.05 DNA copies (p=0.0012). More aggressive proliferation was evident in T cells from cases compared to controls with a 2-fold more rapid decline in TREC copy number each year (0.099 unit decrease in TREC copies per year among cases, p

Description: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis with potential for progression to acute myeloid leukemia (AML). We compared natural killer (NK) cytolytic function in 48 MDS patients with 37 healthy donors and found reduced activity in the patient population (K562 cytolysis, 19% ± 21% SD versus 40% ± 17%) (P 〈 .001). NK cytotoxicity in MDS patients was reduced against 3 disparate tumor targets with differential activating receptor requirement, suggesting global defects in NK function. Reduced NK function in MDS was significantly associated with higher International Prognostic Score (P = .01), abnormal karyotype (P = .05), the presence of excess blasts (P = .01), and age-adjusted bone marrow hypercellularity (P = .04). MDS patients had a display of the activating receptor NKp30, and NKG2D down-regulation closely correlated with impaired NK function (P = .001). NKG2D ligands (MICA and MICB) were expressed on CD34+ cells from bone marrow of 30% of MDS patients and a leukemic cell line derived from an MDS patient (MDS1). Collectively, these findings suggest that impairment of NK cytolytic function derives in part from reduced activating NK receptors such as NKG2D in association with disease progression. Evasion of NK immunosurveillance may have importance for MDS disease progression.

Description: BACKGROUND: A subset of patients with Myelodysplastic Syndrome (MDS) responds well to immunosuppressive therapy (IST) and the only validated predictor of response is age, with younger patients faring much better than older patients. Hematologic improvement on immunosuppressive therapy is associated with a survival benefit with response rates ranging from 15% to 50%, clearly comparable or better than results with other existing therapies in MDS. Despite progress in the basic understanding of immune pathobiology of MDS and a clear therapeutic value, including improved long-term survival, IST including anti-thymocyte globulin (ATG) and/or cyclosporine A (CyA) is rarely offered to MDS patients in the U.S. due to uncertain criteria for selection of patients and potential toxicities. In addition, there is an underlying concern that inappropriate use of immunosuppressive therapy may negatively impact risk for leukemia progression, which occurs in 30–40% of MDS cases. The long-term goal of this study is to identify an immune signature that has postive predictive power for IST responsiveness. METHODS: To determine the effect of age on T-cell homeostasis and function and IST response, we performed a study of 54 MDS patients compared to 37 healthy controls. In a pilot study, T cell abnormalities associated with response to equine anti-lymphocyte globulin (eATG, lymphoglobulin, Pfizer, Inc) and/or CyA was studied in 12 younger MDS patients composed of 6 responders and 6 non-responders. RESULTS: CD4+ T-cells are normally present in the peripheral blood lymphocyte pool at 2 to 4 times greater than that of CD8+ T-cells, and diminished CD4:CD8 ratio has been previously shown to correlate with poor survival outcome in MDS. Similar to previous reports, we found that the age-adjusted CD4:CD8 ratio was reduced in MDS patients compared to healthy controls (p-value

Description: Abstract 1759 Poster Board I-785 Lenalidomide (LEN) is a thalidomide derivative with proven efficacy for the treatment of patients with myelodysplastic syndrome (MDS). High rates of erythroid and cytogenetic response in patients with chromosome 5q deletion [del(5q)] produced the first FDA-approved karyotype-specific treatment for this disease. Transfusion-independence rates of approximately 25% have been reported previously for patients with non-[del(5q)] and efficacy in this population has been linked to the promotion of erythroid differentiation. Because impaired erythroid differentiation in lower-risk MDS may occur through several pathophysiological mechanisms, the identification of additional factors with predictive value for both response and failure to LEN are needed to optimize success of treatment. In addition to affecting erythroid differentiation, LEN has well-known potential for immune modulation and generates highly potent effector T cell responses in vitro and in vivo by potentiating T cell receptor signaling. Immune deregulation mediated by autoreactive effector T cells has been linked to impaired erythropoiesis and granulopoiesis in a distinct subset of MDS patients raising the question of whether LEN impacts the disease process in this subset of patients. To understand the relationship between T cell deregulation and LEN response, we conducted a pilot study of 13 low/INT-1-risk non-del (5q) MDS patients (7 responders and 6 non-responders) treated with LEN and determined 23 covariates related to functional T cell response measured prior to treatment and then correlated to treatment outcome. Of these 23 covariates, multiple T cell immune parameters were analyzed but were not associated with response including interferon-g (IFNg) production by CD4+ T cells (p=0.9) and CD8+ T cells (p=0.27), Tumor Necrosis Factor (TNF)-a production by CD4+ T cells (p=1.0) and CD8+ T cells (p=0.8), TCR-associated proliferation within the CD4+ (p=1.0) and CD8+ (p=0.4) compartment, CD4/CD8 ratio (p=0.3), percentage of CD4+ (p=0.5) and CD8+ (p=0.5) T lymphocytes, and the percentage of naïve and three different types of memory CD4+ T cells. Analysis was performed using two-group comparison statistical tests (two-sample t-test and Wilcoxon rank sum test) to compare responders (R) vs non-responders (NR). Only one factor was independently linked to LEN response. Results showed that a higher percentage of CD8 T cells (mean 56% in NR vs 32% in R) with a Terminal Effector Memory [TEM]) phenotype (CD45RA+/CD62L-) was associated with LEN failure (p=0.02). This population of T cells occurs at a low frequency in healthy individuals but can be induced to differentiate in vitro under constant exposure to long-term antigen and cytokine stimulation. We have shown previously that CD8+ TEM T cells are expanded in patients with impaired myelopoiesis due to immune dysregulation in Large Granular Lymphocyte (LGL) leukemia. In conclusion, these results suggest that CD8+ terminal effector memory expansion may be linked to immune deregulation in MDS and represents an important biomarker with negative predictive importance for LEN response in non-del(5q) low-risk MDS. Disclosures No relevant conflicts of interest to declare.

Description: Background: An increase in CD4+FoxP3+ regulatory T cells (Tregs) has been associated with disease progression in myelodysplastic syndrome (MDS). Tregs provide peripheral tolerance through naturally occurring negative regulatory mechanism that control self-reactivity and autoimmunity. MDS is a heterogeneous hematopoietic disease linked to several distinct pathophysiologic mechanisms that occurs primarily in older individuals. Through the work of our laboratory and others, the balance of CD4 and CD8 T cells has been shown to be disturbed in MDS patients that respond to immunosuppressive therapy. IST offers hematologic benefit and improved survival in a subpopulation of MDS patients with evidence of a T-cell autodominant immune process. It is critical to clarify the correlation between dysregulated naïve and memory CD4+ T cells and dysfunction of Tregs since it has been reported that the supply and function of naïve Tregs is important for protection against autoimmune diseases mediated by T-cells. Methods: We analyzed the direct correlation among FoxP3, CD25 and CD127 expression, and that of CD45RA and CD27 to define Tregs with naïve, central memory, and effector memory phenotypes in 45 MDS patients and 17 healthy controls. We developed an 8-color flow cytometry staining approach that included the following phenotypic markers to define the cell populations: antibodies to CD3, CD4, CD25, CD127 (IL-7Rα), CD45RA, CD27, FoxP3, and aqua fluorescent dye for live:dead cell discrimination. Results: A total of 23 men and 22 women were enrolled from the Bone Marrow Failure Rare Disease Research Network with a diagnosis of MDS with a median age of 69 years old. Based on International Prognostic Scoring System classification, 90% were in a lower risk category (low and intermediate −1 risk). The controls were 17 healthy individuals that donated blood to the Southwest Florida Blood Services (SFBS) and consisted of 8 men and 9 women (median age 52 years old). We found that bright CD25 expression and dim expression of CD127 were most closely associated with FoxP3 expression in healthy controls. The transcription factor FoxP3, which is the most specific phenotypic marker of Tregs, displayed a distinct association with CD25 and CD127 in naïve and memory cells. Therefore, naïve and memory Tregs were defined by FoxP3 expression and the following phenotypes: naïve Tregs (CD45RA+/CD27+/FoxP3+), central memory Tregs (CD45RA−/CD27+/FoxP3+), and effector memory Tregs (CD45RA−/CD27−/FoxP3+). The mean percentage of FoxP3+ Tregs in MDS patients did not differ from healthy controls (5.24% and 4.83%, respectively P=0.94). Younger age, 〈 61 years of age, has been strongly associated with responsiveness to IST and T-cell-mediated autoimmunity. When the Tregs were examined in patients divided into two groups based on an age of 60 years old, a higher percentage of FoxP3 positive cells was observed in the older age group compared to the younger group (mean, 7.46% and 4.98%, respectively, P=0.12) of MDS patients. Interestingly, the percentage of FoxP3+ Tregs tended to be inversely correlated with the lymphocyte count (P=0.08) and the CD4/CD8 ratio (P=0.1) in patients. When naïve and memory Tregs were compared between cases and controls, these Treg populations demonstrated distinct skewing with effector memory Tregs dramatically higher in some patients. MDS patients were then divided into two groups based on the percentage of effector memory Tregs. Using a cutpoint equal to the mean + 2 S.D of controls, the patients were grouped by the percentage of effector memory (EM) Tregs into two groups: higher EM-Tregs (mean 39.3% ± 12.5%) and lower EM-Tregs (mean 6.39% ± 5.16%). The patient group with higher EM-Tregs was significantly associated with the presence of anemia (P=0.01) compared to the group with lower effector memory Tregs Conclusions: Memory Tregs have been reported to express higher levels of integrin alphaE, display differential tissue distribution, and a higher turnover rate compared to naïve Tregs. Memory Tregs differentiate from naïve CD25−FoxP3− non-Tregs in the peripheral blood after antigenic challenge under tolerogenic conditions. Our findings suggest that the conversion of effector memory Tregs may be important during development of anemia in MDS patients with autoimmune-mediated bone marrow failure.

Description: Coagulation proteases are crucial for hemostasis and have also been implicated in inflammatory responses, blood vessel formation, and tumor cell metastasis. Cellular responses triggered by proteases are mediated by protease-activated receptors (PAR). Adeno-associated virus (AAV)-2 vectors hold promise for the treatment of several diseases and were already tested in Phase I studies for hemophilia B following intramuscular or hepatic artery deliveries. Previously, we determined an unexpected inhibitory effect (60–70% downregulation) on AAV-2 and adenovirus mediated gene transfer by thrombin- or FXa inhibitors. These results were independent of mouse strain, transgene product, or vector promoter, and gene expression by vectors of alternate serotypes AAV-5 or -8, which do not share cellular receptors with AAV-2, were not affected by any drug. Here we present in vivo evidence of a novel role of coagulation proteases and PARs in modulating gene transfer by viral vectors. We tested AAV-2 gene transfer efficacy in (a) animal models for proteases deficiency [FX and FIX deficient animals], (b) PAR-1 or PAR-2 deficient mice, (c) and following in vivo activation of PARs. FX knockout mice with residual activity of only 1–3% of normal (n=9) were injected with AAV-2-human(h)FIX vector and compared to littermates with FX levels of 50% (n=4). FIX expression levels were 2-fold lower among FX-deficient mice compared to controls (p