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 ageadjusted 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 ؍ . IntroductionThe myelodysplastic syndromes (MDS) are stem cell malignancies that display hematologic heterogeneity but share features of ineffective hematopoiesis and a potential for progression to acute myeloid leukemia (AML). 1,2 Multiple factors have been implicated in the pathogenesis of MDS, including cytogenetic and molecular abnormalities and disturbance in cellular immunity. Abnormal natural killer (NK) function, including reduced antibody-dependent cell cytotoxicity (ADCC) and diminished direct NK cell cytolytic function, have been previously described; however, the biologic mechanisms underlying these changes have not be defined. [3][4][5][6][7] Normal NK cells, ␥␦ T cells, and some ␣ T cells mediate their biologic action through 3 families of NK receptors: killer cell immunoglobulin-like receptors (KIRs), C lectin-like (NKG2) family receptors, and natural cytotoxicity receptors ([NCRs] eg, NKp30, NKp44, and NKp46). 8 Regulation of innate immunity occurs through balanced signaling by these families of NK receptors with activating and inhibitory function. [9][10][11] Constitutively expressed activating receptors such as NKp46 and NKp30 along with NKG2D mediate most non-major histocompatibility complex (non-MHC)-induced tumor-specific cytotoxicity by NK cells, and the activation-restricted NCR (NKp44) increases NK cytotoxicity after cytokine activation. 12 Although many NK receptors with both activating and inhibitory function have been identified and details of their downstream signaling pathways have been elucidated, a void exists in the identification of activatory NK ligands and the pathologic situations in which they are induced in tumor and virally infected cells. The best-characterized NK receptor ligands are the stress-inducible MHC class I-related chain A (MICA), MICB, and UL16-binding proteins (ULBPs), which constitute the major cellular ligands for human NKG2D. [13][14][15][16][17] In addition to viralinduced expression, NKG2D ligands are often expressed by tumor cells. 18,19 Ligands for NKp30, NKp44, and NKp46 have not yet been delineated.In MDS, the pathogenesis and clinical implications of reduced NK ...
Selected patients with Myelodysplastic Syndromes (MDS) are responsive to immunosuppressive therapy, suggesting that hematopoietic suppressive T cells have a pathogenic role in ineffective hematopoiesis. We assessed T-cell receptor (TCR) clonality through combined flow cytometry and molecular analysis of the complementarity determining region (CDR)-3 of the T-cell receptor-Vb gene. We identified clonal T cells in 50% of MDS patients (n ¼ 52) compared to 5% of age-matched normal controls (n ¼ 20). The presence of T-cell clones was not associated with features linked previously to immunosuppression response, including WHO diagnostic category, karyotype, marrow cellularity, IPSS category, sex or age p60. Using flow cytometry to identify expanded Vb-families, we found that T cells showed greater expansion in the bone marrow compared with peripheral blood, and were characterized as CD8 þ /CD57 þ /CD28 À effector T cells. Expanded effector T cell were CD62L negative and expressed the natural killer C-lectinfamily receptor NKG2D and CD244 (2B4). We conclude that clonal T-cell expansion is common among all MDS prognostic subgroups.
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<0.001). Reduced NK function in MDS was significantly associated with higher IPSS (p=0.01), abnormal karyotype (p=0.05), presence of excess blasts (p=0.01), and age-adjusted bone marrow hypercellularity (p=0.04). MDS patients had reduced display of the activating receptor NKp30, accompanied by NKG2D (an activating C lectin-like (NKG2)-family receptor) downregulation that closely correlated with impaired NK function (p=0.001). NK-mediated cytotoxicity and NKR expression were examined after treatment with lenalidomide 5μM for 72 hours or IL-2 in normal donors and MDS patients. Lenalidomide augmented NK lytic function in normal donors (vehicle, 42% ± 15 vs. lenalidomide, 71% ± 17; P≤0.01 by t test) and in 10 out of 17 (59%) of the MDS patients (vehicle, 25% ± 25 vs. lenalidomide 42% ± 33). IL-2 increased NK cytolytic function in all MDS patients (vehicle, 16% ± 16 vs. lenalidomide, 47% ± 33) and the percentage of NK cells expressing CD69 was increased from both MDS patients and normal controls. Similarly, the percentage of NK cells expressing NKp30, NKG2D, NKp44 but not NKp46 was significantly enhanced by IL-2 culture. In contrast, lenalidomide treatment failed to induce NKR expression in MDS patients and normal controls. Conclusions: These findings suggest that impairment of NK cytolytic function in MDS derives in part from reduced display of the activating NK receptors such as NKG2D, which accompanies disease progression. Lenalidomide augments NK function in some patients; however, the mechanisms of functional augmentation by IL-2 and lenalidomide are divergent. Evasion of NK immunosurveillance may be an important factor associated with MDS disease progression.
INTRODUCTION: Large granular lymphocyte (LGL) leukemia is associated with increased numbers of circulating NK or T-LGL cells with only 5% of these cases related to NK-cell expansion. NK-LGL leukemia is primarily characterized by chronic anemia in association with low to normal numbers of erythroid precursors in the bone marrow and chronic neutropenia. Here, we explore the mechanism of pathogenesis in the first case report of a patient with NK-LGL leukemia in association with primary pulmonary hypertension (PPH). This patient displayed a diffuse pulmonary infiltrate with sustained elevated mean pulmonary artery pressure at rest confirmed by right-sided cardiac catheterization meeting the clinical diagnostic criteria for PPH. Association between T-LGL leukemia and PPH has been reported previously suggesting a causitive link between LGL expansion and PPH pathogenesis. METHODS: Cytotoxicity of a normal pulmonary endothelial cell line (CRL-2598) was determined by 5-hr 51-Cr-release assays using NK-cells from this NK-LGL patient compared to healthy controls. Specific NK receptors were blocked by antibodies added to the effector cells for 30 min prior to the cytotoxicity assay. Production of TNFα was assessed by intracellular cytokine staining with detection by flow cytometry. To block NK receptor signals, dominant negative adaptor proteins, DAP10 and DAP12, were constructed and expressed in recombinant vaccinia viruses. These viruses were engineered to carry a single Y-A mutation at Y102 or a double mutation at both Y9 and Y102 within the YxxM activating ITAM motif of the adaptor protein. Perforin granule mobilization was determined by immunofluorescence microscopy. RESULTS: Sixty-eight % of peripheral blood and bone marrow mononuclear cells from this patient were phenotypically NK-LGLs with skewed reactivity to the anti-CD158a antibody (KIR2DS1/2DS2), which was shown by mRNA analysis to consist primarily of activating KIR2DS1. We examined the susceptibility of CRL-2598 cells to NK lysis and found that patient-derived NK-cells produced greater lysis without prior activation than healthy controls. Furthermore, NK-cells from this patient produced more TNF-α in response to paraformaldehyde-fixed CRL-2598 cells and displayed perforin granule mobilization to the contact site between effector and target cells. Addition of blocking antibodies against the activating receptors NKG2D and KIR2DS1/KIR2DL1 (anti-CD158a) produced a blockade in this process showing that these receptors were responsible for lysis. When DAP10 and DAP12 adaptor signaling steps were blocked, killing and perforin granule redistribution toward the contact site of the conjugated targets was suppressed. CONCLUSION: This study shows that the presence of activating NK-receptors and DAP10/DAP12 adaptors contibuted to lysis of normal endothelial cells in this patient. DAP10 and DAP12 are key initiators for both NK− and T−cell function suggesting that targeted disruption of these signaling events may prove beneficial for autoimmune inflammatory diseases such as PPH and LGL leukemia.
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