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.     

Immunohistochemical localization of progesterone receptor in human decidua of early pregnancy.

Rat monoclonal antibodies to human progesterone receptor were used for immunolocalization studies in human decidua of early pregnancy. Frozen sections of 42 specimens of decidua were stained by the peroxidase-antiperoxidase method (PAP). Progesterone receptor was localized exclusively in the nuclei of decidual and myometrial cells with no specific staining in the cytoplasm. In the decidualized endometrium, stroma were always positively stained. Smooth muscle, pericyte and endothelial cells of blood vessels were extensively stained. Glandular epithelia showed variation in staining, which was positive in the basal but very weak or negative in the superficial layer of the decidua. No specific staining could be detected in the control sections. Of special interest was the positive staining of the endothelium of decidual blood vessels, a finding which has not been reported previously. The cells of the inner lining of vessels that stained with the antiprogesterone receptor antibodies were also Factor VIII positive, thus confirming the endothelial nature of these cells. It is concluded from these results that endothelial cells from human first trimester decidua express progesterone receptors.     

Cytokine production by CD16 CD56bright natural killer cells in the human early pregnancy decidua

Using a cell sorter, CD16^–CD56^bright natural killer (NK)cells were sorted from decidual mononuclear cells at an earlystage of pregnancy. These cells were examined by the reversetranscrlptase-polymerase chain reaction (RT-PCR) method fortheir expression of mRNA coding for the following 12 cytokines:IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), macrophage colonystimulating factor (M-CSF), tumornecrosis factor- (TNF-), interferond- (IFN-), and leukemia inhibitoryfactor (LIF). Although mRNA coding for every cytokine was detectedin decidual mononuclear cells, mRNAs coding for only G-CSF,GM-CSF, M-CSF, TNF-, IFN-, and LIF were detected In CD16^–CD56^brightNK cells. Also, the supernatant of CD16^–CD56^bright NKcell cultures was found to contain G-CSF, GM-CSF, M-CSF, TNF-,IFN-, and LIF. These findings indicate that CD16^–CD56^brightNK cells produce many different cytokines and that these cytokinesmay play an important role in a successful pregnancy.     

Uncoupling activation-induced modulation of CD16 and CD69 in CD56+ cells during AIDS

The immune system of HIV+ patients is chronically activated, which has been associated with a detrimental effect on both innate and acquired immunity during AIDS. We analyzed the expression and modulation of the triggering markers CD69 and CD16 in CD56+ cells from 18 asymptomatic HIV+ individuals and 8 AIDS patients, compared with 21 seronegative subjects. We observed a diminished PMA-induced CD16 downregulation in AIDS patients (p<0.01), associated with low numbers of CD4+ cells (p<0.02). Furthermore, an enhanced unstimulated expression of CD69 in asymptomatic HIV+ patients (p<0.05) was shown. AIDS patients could not efficiently upregulate PHA-dependent CD69 expression (p<0.05), which correlated with low CD4+ counts (p< 0.05). These abnormalities in CD16 and CD69 modulation were recorded in patients under highly active antiretroviral therapy (HAART). Our results demonstrate an altered modulation of two functionally relevant receptors in CD56+ cells from AIDS patients, contributing to our understanding of the immunopathogeny of NK cell dysfunction during disease progression.     

Early changes of affinity and binding sites of progesterone and oestrogen receptors in decidua exposed to RU 486.

Sixty patients with 6-7 weeks of amenorrhoea were randomly allocated to three groups. The women in the first group (control) took a placebo 24 h before undergoing a vacuum aspiration. The patients in the second and third groups were given 200 mg of RU 486 orally, 12 or 24 h before surgical interruption of their pregnancy. Decidua were collected and frozen in liquid nitrogen. By Scatchard plot analysis, the number of cytosolic binding sites (1798 +/- 803 fmol/mg DNA) of progesterone in decidua in the control group was significantly reduced (P less than 0.01) to 696 +/- 408 or 626 +/- 179 fmol/mg DNA by RU 486 treatment for 12 or 24 h respectively. The dissociation constants of both cytosolic and nuclear progesterone receptors in RU 486-exposed decidua were increased (P less than 0.01). The number of nuclear binding sites of oestrogen receptor was significantly higher (P less than 0.05) in decidua with RU 486 treatment for 12 h (178 +/- 77 fmol/mg DNA) compared to the control (89 +/- 32 fmol/mg DNA). The results suggest that RU 486 might regulate progesterone and oestrogen receptors in the decidua of early human pregnancy, either directly or indirectly.     

Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-12

Interleukin (IL)-2 IL-7 and IL-12 stimulate the generation of lymphokine-activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study, we have compared the ability of IL-2, IL-7 and IL-12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL-2 and IL-12 stimulated the CD56⁺ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of soluble TNFR were detected in IL-7-stimulated cultures. The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine-stimulation. Compared to the effects of IL-2, IL-7 induced lower, but substantial levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA, and IL-7 was a more potent GM-CSF-inducing stimulus than IL-12. IL-12 induced higher levels of interferon-γ (IFN-γ) mRNA than did IL-2, and IL-7 only weakly influenced the IFN-γ expression. In accordance with the mRNA studies, IL-7 induced the secretion of high amounts of GM-CSF and no or low levels of IFN-γ, whereas high amounts of IFN-γ and low levels of GM-CSF were detected in supernatants from IL-12-stimulated NK cells. In conclusion, IL-2, IL-7 and IL-12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level.     

The immune potential of decidua-resident CD16+CD56+ NK cells in human pregnancy

Human CD56⁺CD3^? NK cells can be subdivided into two different subsets according to the expression pattern of CD56 and CD16. CD56^+/brightCD16^? (CD16^?) NK cells are prominently cytokine producers with little cytotoxicity whereas CD56^+/dimCD16⁺ (CD16⁺) NK cells are efficient killers with poorer cytokine production potential. In human pregnancy, CD56⁺ decidual (d)NK cells accumulate in the maternal fetal interface to regulate placental immunity and development. Unlike peripheral blood (pb)NK cells, the majority of dNK cells are CD56 positive with limited CD16 reactivity. Our results demonstrated that in normal and pathological pregnancies, CD16⁺ dNK cells are a unique population in comparison to CD16^? dNK subset. The expression of NK activation receptors CD335, CD336, CD244 and CD314 on CD16⁺ dNK subpopulation was lower than that on CD16^? dNK cells. Upon cytokine stimulation with rhIL-12/15/18 or TGFβ blockade, the CD16⁺ dNK subset exhibited more robust response on the expression of IFNG, IL-8 and CD107a, compared to that of the CD16^? dNK subpopulation. Functions of the CD16⁺ dNK subset were shown to be independent of cellular interaction with trophoblast cells. Studies of preeclamptic patients revealed lower proportions of CD16⁺ dNK cells, suggesting potential protective roles of these cells during normal gestations.. Therefore, we suggest that the CD16⁺ dNK subset, through compensating CD16^? dNK cell function, is an indispensable component to regulate decidual immune response and to support placentation.     

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.     

A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells

Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-). However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells. The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.     

Inhibition of anti-GD3-ganglioside antibody-induced proliferation of human CD8+ T cells by CD16+ natural killer cells

The ganglioside GD3 has been described as a membrane component of human T cells which is involved in T cell growth. In the present study the activating function of GD3 for human CD4⁺ and CD8⁺ T cells was analyzed by five different monoclonal antibodies (mAb) directed against the GD3 molecule. Three mAb U5, Z21 and R24 induced strong proliferation of peripheral blood mononuclear cells and purified CD8⁺ and CD4⁺ T cells of normal donors containing less than 5% CD16⁺ natural killer (NK) cells. In contrast to CD4⁺ T cells, CD8⁺ T cells proliferated only weakly in the presence of 15% CD16⁺ NK cells. The proliferative response of purified CD4⁺ and CD8⁺ T cells (<5% NK cells) correlated with the antibody-dependent induction of integral and soluble interleukin-2 (IL-2) receptors and was reduced to 20% by an anti-IL-2 receptor antibody. Our results show, that the GD3 molecule represents an activation molecule for both CD4⁺ and CD8⁺ T cells and that CD16⁺ NK cells selectively inhibit anti-GD3 antibody-induced proliferation of CD8⁺ T cells.     

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.     

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.     

Augmentation by transferrin of IL-2-inducible killer activity and perforin production of human CD8+ T cells.

The effects of human transferrin (Tf) on lymphokine (IL-2)-activated killer (LAK) induction from blood lymphocytes of healthy donors was examined. LAK cells were induced by 6-day incubation in medium with recombinant human IL-2 of lymphocytes, and their cytotoxic activity was assessed by measuring 51Cr release from NK-resistant Daudi cells. Tf alone did not induce any LAK activity, but in combination with IL-2, it augmented LAK induction dose- and time-dependently. This augmenting effect was completely abolished by pretreatment with anti-Tf antiserum. Tf augmented the proliferative response of lymphocytes to IL-2 and their expressions of receptors for IL-2 and Tf. CD8+ T cells were isolated from purified blood lymphocytes using antibody-bound magnetic beads. Addition of Tf to cultures of CD8+ cells resulted in significant augmentation of killer cell induction and perforin (PFP) production after 4 days stimulation with IL-2. These results indicate that Tf is important in generation of IL-2-inducible killer properties and PFP activity of human CD8+ T cells.     

Divergences in KIR2D+ natural killer and KIR2D+CD8+ T-cell reconstitution following liver transplantation

Natural killer (NK) and CD8(+) T cells may be active elements in the allograft response, but little is known about their role in liver transplantation. Some of these cells express killer immunoglobulin-like receptors (KIRs), which after binding specific ligands may transmit inhibitory/activating signals. In this study, circulating NK and CD8(+) T cells expressing CD158a/h (KIR2DL1/S1) or CD158b/j (KIR2DL2/3/S(2)) receptors were analyzed in 142 liver recipients by flow cytometry. They were underrepresented in patients before transplantation, but following transplantation, whereas the KIR2D(+) NK subsets experienced a late recuperation (day 365) mainly in C2-homozygous patients developing early acute rejection, recovery of the 2 CD8(+)KIR2D(+) T cells started earlier, showing significant differences on day 365 between patients without acute rejection and those suffering from it (p = 0.004 and p < 0.0001, respectively). These differences were also evident when the human leukocute antigen-C genotypes of the recipient were considered. In conclusion, whereas the late recovery of KIR2D(+) NK cells in C2/C2 patients appears to be linked to acute rejection, the increase in early CD8(+)KIR2D(+) T cells in overall liver recipients correlates with a most successful early graft outcome. Therefore, monitoring of KIR2D(+) cells appears to be a useful tool for liver transplant follow-up.     

Peripheral natural killer cytotoxicity and CD56(pos)CD16(pos) cells increase during early pregnancy in women with a history of recurrent spontaneous abortion

For diagnostic purposes we assessed peripheral natural killer (NK) cell cytotoxicity and NK and T cell numbers to assess their putative predictive value in recurrent spontaneous abortion (RSA). A total of 43 women with subsequent pregnancy, 37 healthy controls and 39 women successfully partaking in an in-vitro fertilization (IVF) procedure, were included in the study. We show that before pregnancy, levels of NK cytotoxicity and numbers of both single CD56(pos) and double CD56(pos)CD16(pos) cells were similar between RSA women and controls. But notably, within the RSA group, NK cell numbers of <12% were strongly associated with a subsequent pregnancy carried to term. Supplementation of folic acid led to an increase of single CD56(pos) cells, but cytotoxic function appeared unaffected. The expression pattern of killer inhibitory receptors on CD56(pos) cells was not different between patients and controls. A longitudinal study revealed that, compared with controls, in RSA women higher numbers of double CD56(pos)CD16(pos) cells were present during early pregnancy, paralleled by an increase in cytotoxic NK cell reactivity. The single CD56(pos) population decreased in number. In conclusion, the analysis of peripheral NK cell characteristics appears a suitable diagnostic tool in RSA. Immunomodulation aimed at NK cell function appears a promising therapeutic measure.     

Expansion of CD3+CD4-CD8- T cell population expressing high levels of IL-5 in Omenn's syndrome.

Omenn's syndrome is a fatal, autosomal-recessive combined immune deficiency characterized by several erythematous exfoliative eruptions, lymphadenopathy, hepatosplenomegaly, and elevated eosinophil count. In some of these patients an expansion of CD3+CD4-CD8- double negative (DN) T cell population was observed. We show here that the DN population represents a clonal expansion of T cells which preferentially use V beta 14 in their T cell receptor complex. Using polymerase chain reaction, we show that patient's DN cells express spontaneously high levels of IL-5, thus possibly explaining the abundance of eosinophils in this disorder. The increase of IgE observed in patients with Omenn's syndrome is unlikely to be related to IL-4 production, as IL-4 levels in patient samples were low. However, patient's low expression of interferon-gamma (IFN-gamma), which has been reported to inhibit IgE production, may explain the elevated levels of IgE in this patient. The results thus highlight the importance of the inhibitory effect of IFN-gamma on regulation of IgE production.     

Defective expression of high affinity IL-2 receptors on activated T cells from aged humans

The proliferative response of T cells from aged humans to a number of mitogens is significantly reduced. We report here that there is a decrease in high affinity IL-2 receptor (IL-2R) expression on activated T cells from aged humans. Scatchard analysis of the binding of [125I]IL-2 demonstrates fewer high affinity IL-2 binding sites. Autoradiographic techniques demonstrate that this results from there being fewer activated T cells from old as compared to young donors that express high affinity IL-2R. However, T cells from old donors that do not express high affinity IL-2 binding sites express both the IL-2 binding 55 and 75 kd chains. Thus, although the two IL-2 binding peptides are expressed on activated T cells from old donors, expression of the high affinity IL-2R is reduced. This may explain the decreased ability of T cells from old donors to respond to IL-2. The impaired ability of activated T cells from old donors to express high affinity IL-2R while expressing the 55 and 75 kd chains may provide insights into the mechanisms of IL-2 interactions with its receptor.