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.     

Peritoneal natural killer cells from epithelial ovarian cancer patients show an altered phenotype and bind to the tumour marker MUC16 (CA125)

The ovarian tumour marker MUC16 (CA125) inhibits the cytotoxic responses of human natural killer (NK) cells and down-regulates CD16. Here we show that approximately 10% of the peripheral blood NK cells (PBNK) from the epithelial ovarian cancer (EOC) patients are CD16(-) CD56(br) whereas 40% of the peritoneal fluid NK (PFNK) carry this phenotype, which is usually associated with NK cells from the lymph nodes or human decidua. PBNK from healthy donors exposed to PF show a significant increase in the CD16(-) CD56(br) population. This shift in phenotype is not caused by increased apoptosis of the CD16(+) CD56(dim) cells or selective proliferation of the CD16(-) CD56(br) NK cells. Thus, the terminal differentiation of the CD16(-) CD56(br) NK cells to CD16(+) CD56(dim) subset that occurs during normal NK cell development may actually be a reversible step. A majority of the NK cell receptors (NKp46, NKp44, NKG2D, CD244, CD226, CD158a, CD158b, and CD158e) studied were down-regulated in the PFNK. MUC16 binds selectively to 30-40% of CD16(+) CD56(dim) NK cells in EOC patients indicating that phenotypic alterations in these cells are mediated by tumour-derived soluble factors. Similar to EOC, MUC16 in early pregnancy also binds to NK cells suggesting shared mechanisms of NK cell suppression in feto-maternal tolerance and immune evasion by ovarian cancers.     

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.     

Impaired IFN-γ production and proliferation of NK cells in multiple sclerosis

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56(dim)CD16(+)) or immunoregulatory (CD56(bright)CD16(-)) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-γ production of CD56(bright)CD16(-) NK cells but not of their CD56(dim)CD16(+) counterparts were substantially diminished in MS. Impaired expansion of CD56(bright)CD16(-) NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4(+) T cell line CEM and allogeneic primary CD4(+) T-cell blasts was unchanged. Thus, characteristic functions of CD56(bright)CD16(-) NK cells, namely cytokine-induced NK cell expansion and IFN-γ production, are compromised in the NK cell compartment of MS patients.     

Induction of CD16+ CD56bright NK cells with antitumour cytotoxicity not only from CD16- CD56bright NK Cells but also from CD16- CD56dim NK cells

The aim of this study was to examine the effect of cytokines on different subsets of NK cells, while especially focusing on CD16(-) CD56(dim) cells and CD16(-) CD56(bright) cells. When human peripheral blood mononuclear cells (PBMC) were cultured with a combination of IL-2, IL-12 and IL-15 for several days, a minor population of CD56(bright) NK cells expanded up to 15%, and also showed potent cytotoxicities against various cancer cells. Sorting experiments revealed that unconventional CD16(-) CD56(+) NK cells (CD16(-) CD56(dim) NK cells and CD16(-) CD56(bright) NK cells, both of which are less than 1% in PBMC) much more vigorously proliferated after cytokine stimulation, whereas predominant CD16(+) CD56(dim) NK cells proliferated poorly. In addition, many of the resting CD16(-) CD56(bright) NK cells developed into CD16(+) CD56(bright) NK cells, and CD16(-) CD56(dim) NK cells developed into CD16(-) CD56(bright) NK cells and also further into CD16(+) CD56(bright) NK cells by the cytokines. CSFE label experiments further substantiated the proliferation capacity of each subset and the developmental process of CD16(+) CD56(bright) NK cells. Both CD16(-) CD56(dim) NK cells and CD16(-) CD56(bright) NK cells produced large amounts of IFN-gamma and Fas-ligands. The CD16(+) CD56(bright) NK cells showed strong cytotoxicities against not only MHC class I (-) but also MHC class I (+) tumours regardless of their expression of CD94/NKG2A presumably because they expressed NKG2D as well as natural cytotoxicity receptors. The proliferation of CD16(+) CD56(bright) NK cells was also induced when PBMC were stimulated with penicillin-treated Streptococcus pyogenes, thus suggesting their role in tumour immunity and bacterial infections.     

Clinicobiological, immunophenotypic, and molecular characteristics of monoclonal CD56-/+dim chronic natural killer cell large granular lymphocytosis

Indolent natural killer (NK) cell lymphoproliferative disorders include a heterogeneous group of patients in whom persistent expansions of mature, typically CD56(+), NK cells in the absence of any clonal marker are present in the peripheral blood. In the present study we report on the clinical, hematological, immunophenotypic, serological, and molecular features of a series of 26 patients with chronic large granular NK cell lymphocytosis, whose NK cells were either CD56(-) or expressed very low levels of CD56 (CD56(-/+dim) NK cells), in the context of an aberrant activation-related mature phenotype and proved to be monoclonal using the human androgen receptor gene polymerase chain reaction-based assay. As normal CD56(+) NK cells, CD56(-/+dim) NK cells were granzyme B(+), CD3(-), TCRalphabeta/gammadelta(-), CD5(-), CD28(-), CD11a(+bright), CD45RA(+bright), CD122(+), and CD25(-) and they showed variable and heterogeneous expression of both CD8 and CD57. Nevertheless, they displayed several unusual immunophenotypic features. Accordingly, besides being CD56(-/+dim), they were CD11b(-/+dim) (heterogeneous), CD7(-/+dim) (heterogeneous), CD2(+) (homogeneous), CD11c(+bright) (homogeneous), and CD38(-/+dim) (heterogeneous). Moreover, CD56(-/+dim) NK cells heterogeneously expressed HLA-DR. In that concerning the expression of killer receptors, CD56(-/+dim) NK cells showed bright and homogeneous CD94 expression, and dim and heterogeneous reactivity for CD161, whereas CD158a and NKB1 expression was variable. From the functional point of view, CD56(-/+dim) showed a typical Th1 pattern of cytokine production (interferon-gamma(+), tumor necrosis factor-alpha(+)). From the clinical point of view, these patients usually had an indolent clinical course, progression into a massive lymphocytosis with lung infiltration leading to death being observed in only one case. Despite this, they frequently had associated cytopenias as well as neoplastic diseases and/or viral infections. In summary, we describe a unique and homogeneous group of monoclonal chronic large granular NK cell lymphocytosis with an aberrant activation-related CD56(-/+dim)/CD11b(-/+dim) phenotype and an indolent clinical course, whose main clinical features are related to concomitant diseases.     

CD56bright cells differ in their KIR repertoire and cytotoxic features from CD56dim NK cells

In this study we present new differential characteristics of NK cells expressing CD56 surface antigen in low (CD56dim) or high (CD56bright) density. In contrast to CD56bright NK cells CD56dim cells express killer cell immunoglobulin (Ig)-like receptors (KIR) such as CD158a, CD158b, and NKB1. However, c-type lectin-like receptors (KLR) CD94/NKG2 and CD161 are present on both subsets. The ability to form conjugates with susceptible targets is approximately twice as strongly pronounced in CD56dim vs. CD56bright NK cells. Last but not least, granules of CD56dim cells contain about tenfold more perforin and granzyme A enabling potentially more effective cytolysis compared to CD56bright NK cells. On the other hand, CD56bright NK cells are superior in producing the proinflammatory cytokines IFN-gamma (28.5% vs. 20.8%, p<0.05) and TNF-alpha (28% vs. 15.8%, p<0.001). The different NK cell populations retained their specific phenotype in vitro during culture in the presence of IL-2 contradicting that they simply display different stages of maturity. Taken together our data support the view that CD56bright cells are specialized NK cells that regulate immunological response mechanisms rather by cytokine supply than by their cytotoxic potential. The poor cytolytic capacity of CD56bright NK cells can be explained by weak ability in forming conjugates with target cells and low contents of perforin and granzyme A in their granules.     

Characterization of cord blood natural killer and lymphokine activated killer lymphocytes following ex vivo cellular engineering.

Cord blood (CB) natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic cells are poorly characterized but might be used to treat minimal residual and/or recurrent malignant disease. Currently, there is no mechanism to use CB for adoptive cancer cellular immunotherapy after CB transplantation (CBT). Recognizing this as a deficiency, we hypothesized that CB aliquots could be engineered ex vivo for potential donor lymphocyte infusion after CBT. Cryopreserved CB aliquots were thawed, depleted of monocytes, and cultured in serum-free medium alone or serum-free medium with anti-CD3 and interleukins 2, 7, and 12 combined with antibody/cytokines for 48 hours. Immunophenotyping, cytotoxicity, and proliferation were evaluated. A significant expansion of CD3+ was seen, in addition to increases in lymphocyte subsets of CD8+, CD8+/CD25+, and CD3+/45RO+ versus medium alone. A significant enhancement of CD3 proliferation (P<.001), NK cytotoxicity, NK subset expansion, LAK cytotoxicity, and T-helper 1 subset expansion was also demonstrated. Significant enrichment was seen in NK CD16+/CD56+bright, CD16+/CD56+dim, CD56+bright and CD56+dim/KIR3DL1+, CD56+bright and CD56+dim/KIR2DL1+, CD56+bright and CD56+dim/KIR2DL2+ and CD94+/NKG2a+ subsets. These increases in CB NK subsets were in part secondary to augmentation of cell survival. Further, survival of NOD-SCID mice xenografted with human K562 cells and treated with CB cells expanded with antibody/cytokines was significantly higher than that in animals that received no treatment (phosphate buffered saline) and those that were treated with CB ex vivo expanded in medium alone (P<.005, respectively). These data suggest that cryopreserved CB cells could be ex vivo engineered for potential use as adoptive cancer cellular immunotherapy for donor lymphocyte infusion after CBT.     

A novel subset of NK cells expressing high levels of inhibitory FcgammaRIIB modulating antibody-dependent function

NK cells can kill antibody-coated target cells following engagement of FcgammaRIIIA, the major activating FcgammaR expressed by these cells. The presence of FcgammaRIIC (CD32C) has also been reported, but its contribution to the FcgammaR-dependent effector functions of NK cells remains debated. We demonstrate here that inhibitory FcgammaRIIB is also expressed by a small subset of CD56+/NKp46+ NK cells and can efficiently down-modulate their FcgammaR-dependent effector function. Immunofluorescence analyses of NK cells from 52 healthy donors showed the presence of CD56bright/FcgammaRII(-) (5.2%+/-3.4), CD56dim/FcgammaRII(lo/-) (94.1%+/-3.4), and CD56dim/FcgammaRIIbright (0.64%+/-0.72) cells. QRT-PCR and protein analyses performed on isolated FcgammaRIIbright NK cells indicated that FcgammaRIIB is strongly expressed by these cells but not by FcgammaRII(lo/-) cells. In addition, FcgammaRIIbright cells showed a weaker antibody-dependent degranulation when incubated with IgG-coated target cells compared with FcgammaRII(lo/-) NK cells, although a strong FcgammaRIIIA expression was detected in both cells. Furthermore, the addition of anti-FcgammaRII Fab paralleled a higher degranulation of FcgammaRIIbright NK cells, indicating a direct role for FcgammaRIIB in this down-modulating effect. Thus, it is proposed that FcgammaRIIBbright NK cells represent a new NK cell compartment able to down-modulate NK cell functions triggered by the engagement of activating FcgammaR.     

Expression of complement regulatory proteins on human natural killer cell subsets

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely distributed on human leucocytes and protect against complement-mediated damage. To investigate heterogeneity in CReg protein expression by human natural killer (NK) cells, levels were assessed on resting and activated NK cell subsets identified phenotypically on the basis of expression of CD56 and CD158 markers. Levels of all three CReg proteins on CD56+ cells were lower than on T cells (p<0.05). Freshly isolated CD56(bright) cells expressed higher levels of CD55 than CD56dim cells (p<0.05). CD158a+ cells expressed significantly lower levels of both CD46 and CD59, and CD158e+ cells expressed significantly lower levels of CD46, than CD158a(-) CD158e(-) cells, respectively (both p<0.05). Stimulation with PHA did not significantly alter NK cell surface CReg protein levels whereas, following culture with IL-2, CD46 and CD59 were decreased on both CD56bright and CD56dim subsets (p<0.05). In the case of CD59, this was independent of T cells. Only CD46 was significantly downregulated on CD158b+ (GL183+) and CD158e (NKB1+) subsets (p<0.05). However, culture in IL-15 significantly increased levels of all three CReg proteins. These observations that CReg proteins are downregulated on certain NK cell subsets following activation with IL-2 are opposite to previous findings for other leucocyte subpopulations. Activated NK cells may instead use other strategies for protection against complement-mediated damage in a local inflammatory response.     

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.     

Altered Coexpression of Lectin-Like Receptors CD94 and CD161 on NK and T Cells in HIV Patients

HIV patients either on highly active antiviral therapy (HAART) or therapy naive were analyzed for their CD56 phenotype and cytokine production in comparison to healthy controls (HC). Both NK cell populations (CD56(dim) and CD56(bright)) are found to be present in all groups with selectively decreased CD56(dim) NK cells in HAART naive patients. Patients on HAART exhibited significantly diminished numbers of CD161+CD56(dim) NK cells. CD56(dim) were equally potent in producing IFNgamma in all three groups. The number of TNFalpha+CD56(bright) NK cells from patients on HAART and TNFalpha+CD56(dim) NK cells from HAART naive patients was significantly reduced as compared to healthy controls. In summary our data revealed that functional capacities and coexpression patterns of lectin-like receptors on lymphocytes are differentially affected in HIV patients depending on the state of therapy (under HAART or HAART naive) or the cell type (NK or T cells), respectively.     

Differential expression of human granzymes A, B, and K in natural killer cells and during CD8+ T cell differentiation in peripheral blood

NK cells and cytotoxic T lymphocytes can induce apoptosis in virus-infected and transformed target cells via the granule exocytosis pathway. The key components of the cytolytic granules are perforin and several serine esterases, termed granzymes. While the cellular distribution of human granzymes A (GrA) and B (GrB) has been well characterized much less is known about the expression pattern of human granzyme K (GrK). In this study GrA, GrB, and GrK expression was analyzed in human peripheral blood lymphocytes using flow cytometry. There was a distinct population of GrK expressing CD8+ T cells with a CD27+/CD28+/CCR5high/CCR7-/perforin-/low/IFN-gamma+ memory-like phenotype, while all CD56bright NK cells were also positive for GrK. In addition, GrK was also expressed in subpopulations of CD56+ T cells, CD4+ T cells, and TCRgammadelta+ T cells. In contrast, GrB was primarily expressed in CD56dim NK cells and differentiated memory CD8+ T cells with the CD27-/low/CD28-/low/CCR5-/low/CCR7-/CD11b+/perforinhigh phenotype. Only few CD8+ T cells expressed both GrB and GrK. GrA was found to be co-expressed in all GrB- and GrK-expressing T cells. Our findings suggest that granzyme expression during the differentiation process of memory CD8+ T cells might be as follows: GrA+/GrB-/GrK+ --> GrA+/GrB+/GrK+ --> GrA+/GrB+/GrK-.     

G-CSF-mobilized CD34+ cells cultured in interleukin-2 and stem cell factor generate a phenotypically novel monocyte

To study the early stages of development from stem cells of the CD56+ cell population [which includes natural killer (NK) cells], granulocyte-colony stimulating factor-mobilized peripheral blood CD34+ cells from healthy donors were sorted to >99% purity and cultured in the presence of stem cell factor and interleukin (IL)-2. After 3 weeks in culture, the majority of cells acquired CD33, with or without human leukocyte antigen-DR and CD14. In 20 stem cell donors tested, 8.7 +/- 8.8% of cells were CD56+. Two major CD56+ subsets were identified: CD56(bright), mainly CD33- cells (7+/-10%, n=11) with large, granular lymphocyte morphology, and CD56dim, mainly CD33+ (2.5+/-2, n=11) cells with macrophage morphology. The CD56bright population had cytoplasmic granzyme A but lacked killer inhibitory receptor, suggesting they were immature NK cells. The CD56dim, CD33+, population lacked NK markers. They may represent a minor subset of normal monocytes at a developmental stage comparable with the rare CD56+ CD33+ hybrid myeloid/NK cell leukemia. Consistent with a monocyte nature, CD56dimCD33+ proliferated and produced a variety of cytokines upon lipopolysaccharide stimulation, including IL-8, IL-6, monocyte chemoattractant protein-1, and macrophage-derived chemokine but not interferon-gamma. In a short-term cytotoxicity assay, they failed to kill but powerfully inhibited the proliferation of the NK-resistant cell line P815. The generation of CD56+ cells was negatively regulated by hyaluronic acid and IL-4, indicating that extracellular matrix may play an important role in the commitment of CD34+ cells into CD56 myeloid and lymphoid lineages.     

不明原因自然流产与蜕膜NK细胞表面活化性受体表达的相关性研究

探讨不明原因自然流产患者蜕膜NK细胞杀伤活性与其细胞表面活化性受体NKp46、NKp44、NKp30和NKG2D表达的相关性。选取21例早孕不明原因自然流产患者为病例组,25例正常早孕人流妇女为对照组,收集两组的蜕膜组织,Ficoll密度梯度离心分离淋巴细胞,MACS磁珠分选CD3-CD56+NK细胞。以K562细胞为靶细胞,用细胞染色及流式细胞技术检测两组蜕膜NK细胞杀伤活性,用流式细胞技术检测两组蜕膜CD56brightCD16-NK和CD56dimCD16+NK细胞上活化性受体NKp46、NKp44、NKp30和NKG2D的表达,并与NK细胞杀伤活性进行相关性分析。结果:(1)早孕蜕膜NK细胞具有杀伤活性;(2)病例组蜕膜NK细胞的杀伤活性较正常对照组显著增强(P=0.014);(3)病例组蜕膜CD56brightCD16-NK细胞中NKp44的表达比正常对照组显著升高(P=0.021);病例组蜕膜CD56dimCD16+NK细胞中NKp46和NKp44的表达比正常对照组显著升高(分别P=0.026,P=0.041);其余活化性受体的表达两组未见明显差异;(4)蜕膜NK细胞杀伤功能与蜕膜CD56brightCD16-NK细胞中NKp44的表达呈显著正相关(r=0.677,P<0.05),和蜕膜CD56dimCD16+NK细胞中NKp46的表达呈显著正相关(r=0.634,P<0.05)。蜕膜NK细胞活化性受体NKp46和NKp44表达增加,从而使蜕膜NK细胞的杀伤功能增强可能在不明原因自然流产的发病中起重要作用。     

Immunoregulatory factors in multiple sclerosis patients during and after pregnancy: relevance of natural killer cells

Multiple sclerosis (MS) ameliorates typically during pregnancy but after the delivery the relapse rate often increases. Our study was conducted to understand the immunoregulatory mechanisms accompanying this phenomenon. MS patients were followed-up prospectively during pregnancy and 6 months postpartum, with immunological characterization of the peripheral blood. Groups of age- and parity-matched healthy pregnant women, and age- and sex-matched non-pregnant women and non-pregnant MS patients were studied as controls. In our patient cohort, the annualized relapse rate was 1.0 +/- 1.0 relapses/woman/year (mean +/- standard deviation) during the year before pregnancy, but dropped to 0.2 +/- 0.9 during the third trimester (P = 0.02). After the delivery the relapse rate increased again to 1.4 +/- 1.9 (1-3 months postpartum versus third trimester P = 0.003). While percentages of peripheral blood CD3, CD4, CD8 and CD19 immune cell subsets were unchanged over the observation period, reduced disease activity during the last trimester was associated with a significant increase in the percentage of circulating CD56(bright) natural killer (NK) cells. Simultaneously, the proportion of circulating CD56(dim) NK cells was clearly reduced. No alteration was noted in CD4+ CD25(high) forkhead box P3+ regulatory T cells. Production of interferon-gamma by peripheral blood lymphocytes was down-regulated significantly during pregnancy in comparison to the postpartum period, resulting in an increased T helper type 2 (Th2) : Th1 ratio during pregnancy. In conclusion, pregnant state in MS patients is characterized by an increase in the percentage of CD56(bright) NK cells and by enhanced Th2 type cytokine secretion. Our findings suggest a potential role for CD56(bright) regulatory NK cells in the control of autoimmune inflammation during pregnancy in MS.     

Interleukin-1beta costimulates interferon-gamma production by human natural killer cells

Natural killer (NK) cells are an early source of immunoregulatory cytokines during the innate immune response to viruses, bacteria, and parasites. NK cells provide requisite IFN-gamma to monocytes for the elimination of obligate intracellular pathogens. IL-1beta is a pro-inflammatory cytokine produced by monocytes (i.e. a monokine) during the early immune response to infection, but its role in promoting human NK cell IFN-gamma production is unknown. The current study examines the ability of the monokine IL-1beta, plus IL-12, to costimulate IFN-gamma production by resting CD56(bright) and CD56(dim) human NK cell subsets. CD56(bright) NK cells stimulated with IL-1beta plus IL-12 produced abundant IFN-gamma protein, while little IFN-gamma was produced in identical cultures of CD56(dim) cells. In addition, upon activation with IL-1beta, CD56(bright) NK cells exhibited considerably greater phosphorylation of extracellular signal-regulated kinases p42/44 as compared to CD56(dim) NK cells. Quantitative PCR analysis showed brisk induction of IFN-gamma gene expression following costimulation with IL-1beta plus IL-12 in CD56(bright) NK cells, but intracellular flow cytometry revealed that only a fraction (42+/-2.3%) of CD56(bright) NK cells account for this high IFN-gamma production. These data suggest that the monokine IL-1beta is a potent costimulus of IFN-gamma production by a subset of NK cells following infectious insult.     

Differential responses to interleukin 2 define functionally distinct subsets of human natural killer cells.

Human natural killer (NK) cells can be subdivided into two populations based on the density of cell surface CD56 antigen. The great majority (approximately 90%) of NK cells express CD56 at low levels (the CD56dim phenotype), whereas a small NK cell subset (approximately 10%) exhibits approximately fivefold greater density of surface CD56. Exposure to exogenous interleukin 2 (IL 2) induces tenfold greater proliferation of CD56bright cells compared to CD56dim lymphocytes, even though both subsets constitutively express similar levels of intermediate affinity IL 2 receptor (IL 2R) p75 chains. Incubation with IL 2 alone or irradiated target cells alone could induce expression of the IL 2R p55 chain by both CD56bright and CD56dim NK cells; a combination of both stimuli was most effective. IL 2R p55 induction was evident after co-culture of NK cells with both NK-sensitive and NK-resistant cell lines or with antibody-coated target cells. Activation of NK cells with IL 2 plus target cells resulted in enhanced proliferation compared to activation with IL 2 alone; target cells alone did not induce significant proliferation. Although both NK cell subsets appeared to express high-affinity IL 2R p75/p55 heterodimers after stimulation with target cells and IL 2, proliferation of CD56dim cells remained minimal after such activation; activated CD56dim cells consistently demonstrated less proliferation to IL 2 than did resting CD56bright cells. In contrast, CD56bright NK cells exhibited even greater proliferation after stimulation with target cells. Almost all CD56dim NK cells expressed CD16 (Fc gamma R III) as well as the NK zeta chain, whereas less than 50% of CD56bright cells express either CD16 or zeta. CD56bright and CD56dim lymphocytes, thus, appear to represent distinct subpopulations of NK cells with different functional activities. Unlike CD56bright cells, CD56dim NK cells do not proliferate optimally to IL 2, even after the latter have been stimulated to express both IL 2R p55 and IL 2R p75. Efficient proliferation of CD56dim NK cells may, thus, require additional or alternative signals.     

Immunophenotype of peripheral T lymphocytes, NK cells and expression of CD69 activation marker in patients with recurrent spontaneous abortions, during the mid-luteal phase

PROBLEM: Determination of subpopulations of T lymphocytes, natural killer (NK) and activation status, in peripheral blood during the mid-luteal phase from patients with unexplained recurrent spontaneous abortion (RSA). METHOD OF STUDY: Peripheral blood samples from non-pregnant women with RSA and normal multiparous were taken and evaluated for subpopulations of T lymphocytes: CD4, CD8, ('naive-like' and 'memory-like'), TCR receptor (alphabeta and gammadelta), activation status by CD69(+surface or intracellular)/CD3(+), and NK cells (CD16(+)/CD56(dim)/CD3(-), CD16(+)/CD56 (bright)/CD3(-), CD69(+surface or intracellular)/CD56(+)/CD3(-) cells). RESULTS: The evaluation of T lymphocytes only showed an increase in the expression of CD69 (surface and intracellular) in the RSA group. Additionally, we observed an increase in the total NK cells, CD56(+) NK cells percentages, CD56(dim) NK cells and CD69 NK cells in RSA group. CONCLUSION: These observations support the concept that immunological activation of T lymphocytes and NK cells could be involved in peripheral blood during the mid-luteal phase in patients with unexplained RSA.     

An increase in the absolute count of CD56dimCD16+CD69+ NK cells in the peripheral blood is associated with a poorer IVF treatment and pregnancy outcome

BACKGROUND: Our aim was to evaluate the effect of the absolute count of the activation marker (CD69), IgG Fc receptor (CD16) and inhibitor marker (CD94) expression on peripheral blood natural killer (NK) cells on implantation and miscarriage rates after IVF treatment. METHODS: Prospective observational study of 138 randomly selected women who underwent IVF treatment from December 2002 to September 2003. NK cells were identified as CD56(+) (dim + bright) and CD3(-) by flow cytometry. The absolute counts of the CD69(+), CD16(+) and CD94(+)expressing NK cells were recorded and their relation to IVF treatment outcome and miscarriage rate was analysed. RESULTS: The mean (+/-SD) absolute count of the CD56(dim)CD16(+)CD69(+) NK cells for women who had a successful ongoing pregnancy was 0.61 x 10(6)/l (+/-0.31). For those women who failed to achieve a pregnancy, the mean value of the absolute count of CD56(dim)CD16(+)D69(+) NK cells was significantly (P=0.003) higher at 1.66 x 10(6)/l (+/-0.52). The absolute count of CD56(dim)CD16(+)CD94(+) and CD56(dim)CD16(+) NK cells did not show any statistically significant differences between those women with successful and failed IVF treatment. Receiver operating characteristic (ROC) curve analysis was performed to select a CD69 threshold for further statistical analysis. The implantation rate (IR) was significantly lower (13.1%) and miscarriage rate (MR) was significantly higher (66.7%) for women with an absolute CD56(dim)CD16(+)CD69(+) NK cell count of >1.0 x 10(6)/l compared to women with count below this value (IR 28.2% and MR 16.7%). Further analysis of the absolute count of CD56(bright)CD69(+) and CD56(bright)CD94(+) NK cells did not show any significant difference between those women with successful and failed IVF treatment. CONCLUSIONS: An increase in the absolute count of activated NK cells (CD56(dim)CD16(+)CD69(+)) in the peripheral blood is associated with a reduced rate of embryo implantation in IVF treatment. Furthermore, women with high CD56(dim)CD16(+)CD69(+) peripheral blood NK cell absolute count, who are able to achieve pregnancy, have a significantly higher miscarriage rate.