Preferential apoptosis of CD56dim natural killer cell subset in patients with cancer

Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.     

Diffuse large B cell lymphoma expressing the natural killer cell marker CD56

The expression of the natural killer (NK) cell antigen, CD56, in hematological malignancies is rare. However, there are several reports that some hematological malignancies, such as T/NK cell lymphoma, multiple myeloma (MM) and acute myeloid leukemia (AML), express this molecule. In B cell non-Hodgkin's lymphomas (NHL), however, very limited number of cases have been reported to express CD56 molecule. Although one study has recently described that half of microvillous B cell lymphoma (MVL), an uncommon subset of large cell lymphoma, expressed CD56, there have been no reports about most common type of B-NHL, diffuse large B cell lymphoma (DLBL) other than a mention of weak CD56 expression in one of 83 DLBL. We herein presented the first case of diffuse large B cell lymphoma expressing CD56 clearly. The immunophenotype determined by immunostaining and flow cytometric analysis was CD10+, CD19+, CD20+, CD45RO-, CD3- and CD56+. On immunohistochemical study, neither bcl-2 nor TIA-1 was positive for tumor cell. Monoclonal immunoglobulin heavy chain (IgH) gene rearrangement was detected, and the sequence analysis of the variable region of IgH (VH) suggested that this tumor was derived from antigen selected post germinal center B cell. Conventional combination chemotherapy (CHOP) was administered, and the patient has still been in complete remission for 10 months.     

Large natural killer cell lymphoma arising from an indolent natural killer cell large granular lymphocyte proliferation

Natural killer cell large granular lymphocyte proliferation is a relatively rare disorder that typically runs a chronic, indolent course. We present a patient with a 3 1/2-year history of natural killer cell large granular lymphocyte proliferation terminating in large cell lymphoma with natural killer cell features. The diagnosis of natural killer cell large granular lymphocyte proliferation was based on flow cytometric demonstration of an expanded population of CD3- CD16+/CD56+ lymphocytes in the peripheral blood. The patient experienced various rheumatologic symptoms, but was hematologically stable for 3 1/2 years. He then developed fevers, night sweats, weight loss, and a left lower lobe lung mass. Resection of the mass showed a large cell lymphoma with immunohistochemical positivity for CD2, CD7, CD56, and T-cell intracellular antigen-1, compatible with natural killer cell origin. In situ hybridization for Epstein-Barr virus and polymerase chain reaction analysis for T-cell receptor gene rearrangement were negative. To our knowledge, this is the second documented report of chronic natural killer cell large granular lymphocyte proliferation terminating in an aggressive large natural killer cell lymphoma.     

Dissociation of natural killer and lymphocyte-activated killer cell lytic activities in human CD3− large granular lymphocytes

CD3^? large granular lymphocytes (LGL) are known to display natural killer cell (NK) activity without prior sensitization or restriction by major histocompatibility antigens. Upon short-term exposure to interleukin-2, NK cells were shown to acquire lymphocyte-activated killer cell (LAK) activity. The aim of this study was to analyze the characteristics of these lytic activities. Our data indicated that both NK and LAK activities were Ca²⁺ dependent; however, they could be dissociated by a Ca²⁺ channel blocker or a Ca²⁺ channel competitor agent. Moreover, NK activity was associated with granule exocytosis of lytic proteins spontaneously present in CD3^? LGL, the most likely candidate being the pore-forming protein perforin. By contrast, LAK activity was found to be dependent on de novo protein synthesis and distinct from granule exocytosis. Our results strongly suggest that NK and LAK activities could be defined as two distinct pathways involving different lytic mediators.     

Leukaemia of natural killer cell large granular lymphocyte type with HLA-DR-CD16-CD56bright+ phenotype.

The case is reported of a 45 year old woman with the rare leukaemia of natural killer cell large granular lymphocyte (NK/ LGL) type. Cytometric analysis of leukaemic blasts showed that they were positive for CD2, CD38, and CD56 antigens but negative for a series of antigens including CD3, CD7, CD16, and HLA-DR. Rearrangements of the beta T cell receptor, and heavy and kappa immunoglobulin genes were not detected and neither were chromosomal abnormalities. Leukaemic blasts developed NK cytotoxicity. The patient failed to respond to aggressive chemotherapy and died three months after diagnosis. The lack of expression of HLA-DR is an extraordinary characteristic of this case, as all cases of acute NK cell leukaemias described to date expressed HLA-DR. The immunophenotype observed in the NK cell leukaemic blasts may represent the counterpart of a hypothetical normal cell precursor in an early stage of ontogenic NK cell development.     

Permanent large granular lymphocytosis in the blood of splenectomized individuals without concomitant increase of in vitro natural killer cell cytotoxicity.

Increased numbers of circulating lymphocytes and large granular lymphocytes (LGL) were observed in 115 individuals splenectomized for haematological disease (74 cases) or for trauma (41 cases). LGL lymphocytosis was present in 78.4% of haematologically indicated and in 85.4% of traumatic splenectomies. A 70.5% of these values was above the 97.5 percentile upper tolerance limit of healthy controls (200 cases). In addition, 175 haematological controls were investigated. Forty per cent or more of circulating lymphocytes exhibited LGL morphology in nearly half of repeatedly investigated splenectomized persons. The increase in LGL is not attributable to lymphocytosis. It becomes apparent, and persists after the first postoperative week, irrespective of the cause of surgery. In spite of the two-fold increase in LGL concentration in the blood, in vitro natural killer (NK) and antibody dependent cellular cytotoxic (ADCC) activities did not increase in the investigated 48 (NK) and 31 (ADCC) splenectomized persons, as compared with the appropriate healthy or haematological controls.     

Proliferative and cytotoxic capabilities of CD16+CD56- and CD16+/-CD56+ natural killer cells

Natural killer (NK) cells can be divided into several subpopulations according to their expression of the surface antigens CD16 and CD56. The modest quantity of NK cells in the blood available for functional analysis has been a limitation in studies of NK cell subpopulations. In the present study, epinephrine infusion was used to induce lymphocytosis before immunomagnetic methods were applied to isolate CD16+/-CD56+ and CD16+CD56- CD3- NK cells. These subpopulations were compared according to their proliferative and cytotoxic capabilities in 10 human immunodeficiency virus (HIV)-infected individuals and 5 healthy controls. The CD16+CD56- NK cell subgroup had a higher proliferative capacity, whereas the CD16+/-CD56+ NK cell subgroup was mainly cytotoxic, and unaffected by HIV serostatus. This study thus suggests that NK cell phenotypes more strongly predict NK cell function than HIV serostatus. This assertion should be considered when studying NK cell function in subjects with a deviating composition of NK cells.     

Decreased natural killer (NK) cell function in chronic NK cell lymphocytosis associated with decreased surface expression of CD11b

Chronic natural killer cell lymphocytosis (CNKL) is characterized by greatly increased numbers of natural killer (NK) cells and patients with this disease may survive for long periods. This is in contrast to patients with leukemic proliferations of NK cells who can have a rapidly progressive clinical course. We identified a pediatric patient who was largely healthy who had CNKL and we sought to determine if the expanded CD16(+)CD3(-) population in this patient functions differently than classical NK cells. Cytotoxic activity against NK cell-sensitive K562 target cells was present, but lower than that in control donors when calculated as lytic units per CD16(+)CD3(-) cell. This cytolytic activity was inducible in patient samples by IL-2/IL-12 stimulation proportionately to that induced in samples from control donors. Intracellular perforin was also present and induced in patient CD16(+)CD3(-) cells similarly to controls. Other presumed NK cell activities, such as IL-2/IL-12 induced IFN-gamma expression and initiation of apoptosis evidenced by annexin V binding after CD16 crosslinking were present in patient samples. Patient CD16(+)CD3(-) cells, however, differed from classical NK cells, as the majority did not express CD56, CD57, CD8, or CD11b. Most convincingly, there was a 5 log decrease in CD11b expression in patient CD16(+)CD3(-) cells compared to control as determined by mean channel fluorescence. These observed differences may explain the relatively benign phenotype of this disorder.     

Functional and Vbeta repertoire characterization of human CD8+ T-cell subsets with natural killer cell markers,CD56+ CD57- T cells,CD56+ CD57+ T cells and CD56- CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK-type T cell); CD56 single positive (CD56)-T cells, CD56/CD57 double positive (DP)-T cells and CD57 single positive (CD57)-T cells in the peripheral blood. All NK-type T-cell populations expressed CD122 and intermediate levels of T-cell receptor (TCR; regular CD8+ T cells are CD122- and express high levels of TCR). The number of both DP-T cells and CD57-T cells, but not CD56-T cells, gradually increased with age. All NK-type T-cell populations produced larger amounts of interferon-gamma than did regular CD8+ T cells after stimulation with interleukin (IL)-2, IL-12 and IL-15. However, CD56-T cells and CD57-T cells but not DP-T cells showed a potent antitumour cytotoxity to NK-sensitive K562 cells, whereas only CD56-T cells showed a potent cytotoxity to NK-resistant Raji cells. Furthermore, although NK-type T cells produced large amounts of soluble Fas-ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VbetaT cells were found in each NK-type T-cell population. The non-variant CDR3 region(s) for the TCRbeta chain(s) showed CD57-T cells and CD56-T cells to be derived from distinct origins, while the DP-T cell population consisted of a mixture of the clones seen in both CD56-T cells and CD57-T cells. Our results suggest that CD57-T cells and CD56-T cells are functionally and ontogenically different populations while DP-T cells appear to originate from both CD56-T cells and CD57-T cells.     

Altered distribution of natural killer cell subsets identified by CD56, CD27 and CD70 in primary and chronic human immunodeficiency virus-1 infection

Human natural killer (NK) (CD3- CD56+) cells can be divided into two functionally distinct subsets, CD3- CD56(dim) and CD3- CD56(bright). We analysed the distribution of NK cell subsets in primary and chronic human immunodeficiency virus-1 (HIV-1) infection, to determine if HIV infection stage may influence the subset distribution. In primary infection, contrary to chronic infection, the CD3- CD56(dim) subset was expanded compared to healthy controls. We also studied the effect of antiretroviral therapy administered early in infection and found that NK cell subset distribution was partially restored after 6 months of antiretroviral therapy in primary infection, but not normalized. Recently, NK cells have been divided into CD27- and CD27+ subsets with different migratory and functional capacity and CD27-mediated NK cell activation has been described in mice. We therefore investigated whether CD27 and/or CD70 (CD27 ligand) expression on NK cells, and thus the distribution of these novel NK subsets, was altered in HIV-1-infected patients. We found up-regulated expression of both CD27 and CD70 on NK cells of patients, resulting in higher proportions of CD27(high) and CD70(high) NK cells, and this phenomenon was more pronounced in chronic infection. Experiments conducted in vitro suggest that the high interleukin-7 levels found during HIV-1 infection may participate in up-regulation of CD70 on NK cell subsets. Imbalance of NK cell subsets and up-regulated expression of CD27 and CD70 initiated early in HIV-1 infection may indicate NK cell activation and intrinsic defects initiated by HIV-1 to disarm the innate immune response to the virus.     

Immunology: Distribution of cell adhesion molecules on CD56++, CD3-, CD16- large granular lymphocytes and endothelial cells in first-trimester human decidua

Human decidua exhibits a unique infiltrate of large granularlymphocytes (LGL) with a natural killer (NK) cell phenotype(CD56++, CD16–, CD3–). The mechanisms underlyingthe binding of circulating LGL to vascular endothelium in thedecidua and their migration into the decidual stroma were investigatedimmunohistochemically in first-trimester decidua with antibodiesagainst endothelial adhesion molecules and their counter-receptorson leukocytes. Decidual and peripheral blood LGL were also investigatedby flow cytometry. In the immunohistochemical investigations,moderate to large numbers of lymphoid cells in the decidua werefound to express the ₄ and _L integrin subunits, platelet endothelialcell adhesion molecule (PECAM) and intercellular adhesion molecule-1(ICAM-1). PECAM and ICAM-1 were found on the endothelium oflarge numbers of decidual blood vessels of all types. Vascularcell adhesion molecule (VCAM), however, was found on the endotheliumof only small to moderate numbers of arterioles and venulesand a few capillaries, the latter being the main site of migrationof leukocytes into the stroma. Weak staining for endothelialleukocyte adhesion molecule (ELAM) was seen only in a moderatenumber of blood vessels. Flow cytometry revealed expressionof the _L integrin subunit by 72 ± 10% and 97 ±3% of decidual and peripheral blood CD56+ LGL, respectively,of the 4 integrin subunit by 85 ± 7% and 90 ±5%, of PECAM by 40 ± 12% and 30 ± 15%, and ofICAM-1 by 22 ± 10% and 1 ± 1%. These findingssuggest that interaction between the integrin _2L and ICAM-1is the more important mechanism of binding to endothelium inthe migration of CD56+ LGL out of the peripheral blood intothe decidua.     

Morphological, phenotypic and functional characteristics of a pure population of CD56+ CD16- CD3- large granular lymphocytes generated from human duodenal mucosa.

Interleukin-2 (IL-2)-dependent large granular lymphocytes (LGL) with a distinctive surface phenotype were generated from histologically normal duodenal biopsy tissues. Immunoperoxidase staining of the mucosa with an anti-CD56 monoclonal antibody revealed LGL localized in the lamina propria rather than in the epithelium. Light and electron microscopy demonstrated azurophilic and electron-dense cytoplasmic granules. Flow cytometry analysis revealed that these cells express CD45, CD56, CD2, CD7, CD11a, CD18, CD69 and the intermediate affinity (p70) IL-2 receptor (IL-2R) but not CD57, CD16, CD3, CD4, CD5, CD8, CD45RA, CD25, or the high affinity p55 IL-2R. The LGL proliferated when cultured in the presence of human rIL-2 but not in the presence of human rIL-4. Functional studies demonstrated that the LGL had strong cytotoxicity against natural killer (NK) target cells, K562, but not NK-resistant targets such as Colo 205, Melanoma and Epstein-Barr virus (EBV)-transformed B-cell lines. The LGL expressed genes for IL-5, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha) and the corresponding cytokines were detected in culture supernatant. These results provide evidence for an important role of gut mucosal LGL in the induction and regulation of inflammation and immunity in the gut.     

Histopathologic and molecular aspects of CD56+ natural killer/ T-cell lymphoma of the testis

Primary nasal-type natural killer/T-cell lymphoma of the testis is a rare malignancy. Although dissemination to the testis from other sites occurs somewhat more frequently than a primary presentation, even secondary testicular involvement is uncommon. In this article, the authors report on the comprehensive histopathologic, immunohistochemical, and molecular analysis of a case of primary testicular nasal-type natural killer/T-cell lymphoma, and review the features of 16 previously reported patients. The investigation carried out in this study indicates that the testicular nasal-type natural killer/T-cell lymphomas occur at a younger age than their B-cell counterparts, express cytoplasmic CD3 and surface CD56, and consistently show an infection by Epstein-Barr virus. These tumors have variable expression of T-cell antigens other than cytoplasmic CD3 and may show monoclonal rearrangement of T-cell receptor genes. Testicular natural killer/T-cell lymphomas of nasal type invariably follow an aggressive clinical course.     

乙型肝炎病毒导致慢性乙型肝炎患者血中CD56dimNK细胞亚群数量下降

目的观察乙型肝炎病毒对慢性乙型肝炎患者NK细胞亚群的影响。方法利用流式细胞术检测20例正常人及62例慢性乙型肝炎患者人外周血淋巴细胞CD3、CD4、CD8、CD56、CD16的表达;同时观察NK细胞亚群改变与乙型肝炎病毒携带量的相关性。结果①人类NK细胞根据CD56分子的表面密度可分为两个截然不同的群体,绝大多数（约90％）的NK细胞低水平表达CD56（CD56^dim）且高表达CD16,少数NK细胞（10％）高水平表达CD56（CD56^bright）且缺乏或低水平表达CD16;②慢性乙型肝炎患者CD56^brightNK细胞的表达及绝对数量较正常对照组无明显的改变,CD3^-CD56^＋、CD3^-CD56^dimNK细胞的绝对数量较正常对照组显著减少（P〈0．05）;③慢性乙型肝炎患者NK细胞亚群改变与患者的病毒携带量无关;④慢性乙型肝炎患者CD3^＋、CD3^＋CD4^＋比例及绝对数量较正常对照组显著减少（P〈0．05）。结论机体感染乙型肝炎病毒后,导致CD56^dim增殖的关键因子IL-2、IL-21活性下降,使CD56^dim数量选择性减少,减弱NK细胞介导机体ADCC的功能。     

Natural killer cell receptor expression reflects the role of human cytomegalovirus in the pathogenesis of a subset of CD4+ T-cell large granular lymphocytosis

A high frequency of CD4(+) T-cell large granular lymphocyte (T-LGL) lymphocytosis occurs in human leukocyte antigen (HLA) -DRB1*0701 individuals displaying monoclonal expansions of Vβ13.1+ CD4(+) T-cell clones, which specifically respond to human cytomegalovirus (HCMV) antigens. We previously reported the expression of natural killer (NK)-cell associated receptors (NKR) by HCMV-specific cytolytic CD4(+) T cells from healthy donors. In the present study a high expression of different NKR (i.e., NKG2D, killer Ig-like receptors (KIR), CD94, ILT2) was observed in CD4(+) T cells from both Vβ13.1- and Vβ13.1+ CD4(+) T-LGL cases. Remarkably, elevated numbers of CD94/NKG2C+ NK cells, previously shown to expand in association to HCMV infection, were preferentially found in Vβ13.1+ T-LGL, further supporting its role in the pathogenesis of a subset of CD4(+) T-LGL.     

Cell populations in the human early pregnancy decidua: natural killer activity and response to interleukin-2 of CD56-positive large granular lymphocytes.

Large granular lymphocytes (LGL) have been shown previously to be the most abundant cell type in the first trimester human decidua. Purified populations of decidual LGL were prepared by flow cytometry of cell dispersions labelled with NKH1 (CD56), an antibody specific for peripheral blood LGL, and the functional properties of CD56-positive cells, CD56-negative and unsorted decidual cells compared. Both CD56-positive cells and unsorted decidual cells have cytotoxic activity against the natural killer (NK) cell target K562 which was weak compared with that of peripheral blood mononuclear cells (PBMC). The CD56-negative cells had no cytotoxic activity against K562. All three decidual cell populations proliferated in response to recombinant human interleukin-2 (rIL-2), but none produced detectable levels of IL-2 in culture. When unsorted decidual cells were cultured for 7 days in rIL-2 the proportion of CD56-positive cells increased and NK activity against K562 was augmented. The NK activity of purified CD56-positive decidual cells was also augmented by culturing in rIL-2. The potential role of decidual LGL in regulating the development of the semi-allogeneic placenta is discussed.     

Bcl-xL is associated with the anti-apoptotic effect of IL-15 on the survival of CD56(dim) natural killer cells

Human NK cells can be distinguished into CD56(bright) and CD56(dim) subsets based on cell surface CD56 density. It has been shown that IL-2 and IL-15 have opposing effects on life and death of CD8(+) T cells. However, the roles of IL-2 and IL-15 in regulating these two NK cell subsets remain elusive. In this study, we comparatively analyzed the effects of IL-2 and IL-15 on two NK cell subsets. IL-15 improved the proliferation and activation of CD56(dim) NK cells in long-term cord blood mononuclear cell culture, but IL-2 only maintained the survival of CD56(bright) NK cells. The percentage of CD56(+)Annexin V(+) NK cells cultured with IL-15 was lower than that with IL-2; moreover, most of Annexin V(+) NK cells were primarily in the CD56(dim) NK cells. IL-15 cultured NK cells expressed higher level of Bcl-xL than IL-2 cultured cells. Furthermore, IL-15 more strongly upregulated CD25 expression and better maintained the expression of IL-15Ralpha than IL-2. These results suggest that CD56(dim) NK cells undergo apoptosis when cultured with IL-2, but IL-15 inhibits their apoptosis and Bcl-xL is associated with the anti-apoptotic effect of IL-15. So IL-15 played a crucial role in sustaining long-lasting functions of CD56(dim) NK cells.     

CD56(+dim) and CD56(+bright) cell activation and apoptosis in hepatitis C virus infection

CD3- CD56(+dim) natural killer (NK) cells, which are cytotoxic against virally infected cells, may be important in hepatitis C virus (HCV)-infected patients who are successfully treated with pegylated interferon (PEG-IFN)-alpha. We used flow cytometry to enumerate activated (CD69+) and apoptotic (annexin-V+) dim (CD3- CD56(+dim)) and bright (CD3- CD56(+bright)) NK cells obtained from HCV-infected patients before treatment (n=16) and healthy controls (n=15) in the absence and presence of pegylated interferon (PEG-IFN)-alpha-2b. A subset of HCV-infected patients, subsequently treated with PEG-IFN-alpha-2b in vivo, was determined to have a sustained virological response (SVR, n=6) or to not respond (NR) to treatment (n=5). In the absence of IFN, activated dim (CD3- CD56(+dim) CD69+) NK cells were significantly decreased (P=0.04) while activated apoptotic dim (CD3- CD56(+dim)CD69+ annexin-V+) NK cells tended to be increased (P=0.07) in SVR patients compared with NR patients. Activated bright (CD3-CD56(+bright)CD69+) and activated apoptotic bright (CD3- CD56(+bright)CD69+ annexin-V+) NK cells were significantly correlated (P=0.02 and P=0.01, respectively) with increasing hepatic inflammation. These findings suggest that in the absence of PEG-IFN, activated dim (CD3- CD56(+dim)CD69+) NK cell turnover may be enhanced in SVR compared with NR patients and that activated bright (CD3- CD56(+bright)CD69+) NK cells may play a role in liver inflammation.     

Blastic natural killer (NK) cell leukemia (agranular CD4+CD56+ leukemia)

Blastic NK cell lymphoma is a rare hematolymphoid neoplasm. This report illustrates an unusual presentation of this entity, namely as a primary leukemia, but without skin lesions.