Elevated Peripheral Blood Natural Killer Cells Are Effectively Downregulated by Immunoglobulin G Infusion in Women With Recurrent Spontaneous Abortions

PROBLEM: We investigated the hypothesis that elevated peripheral blood natural killer cells (NK) are decreased by immunoglobulin G infusion (IVIg) therapy in women with recurrent spontaneous abortions (RSA) and elevated NK cells. METHODS: Seventy-three women with RSA and elevated NK cells received IVIg therapy (400 mg/Kg/day for 3 days ever 4 wks) and anticoagulation treatment. Peripheral blood immunophenotype assay by flow cytometry was done prospectively prior to and 7 days after first IVIg therapy, every 2 wks until 20 wks gestation and then monthly. Controls were 95 women with RSA and normal NK cells who received anticoagulation treatment. RESULTS: (1) 86.3% of women with elevated NK cells who received the IVIg and anticoagulation therapy had a successful pregnancy outcome; (2) Peripheral blood CD56+ NK cells and CD56+/16+ NK cells were significantly suppressed 7 days post IVIg infusion (P < 0.0005); (3) Pre-IVIg infusion levels of other lymphocyte subsets were not different as compared with those of 7 days post-IVIg therapy; (4) Women who delivered a liveborn infant with IVIg therapy demonstrated downregulation of peripheral blood NK cells (CD56+, CD56+/16+) during early pregnancy when compared to women who miscarried the index pregnancy (P < 0.05); (5) Women with normal NK cells who miscarried while on anticoagulation therapy demonstrated significantly elevated CD56+ NK cells during early pregnancy as compared with that of women who delivered a liveborn infant (P < 0.05); (6) CD19+ B cells were significantly downregulated during pregnancy in women with anticoagulation and IVIg therapy when compared to women with anticoagulation therapy (P < 0.05). CONCLUSION: Downregulation of NK cells in women with RSA is associated with a favorable pregnancy outcome. Peripheral blood NK cells (CD56+, CD56+/16+) are effectively suppressed after IVIg therapy. Women with RSA and high NK cells benefit from IVIg therapy and experience suppression of CD56+ and CD56+/16+ NK cells.     

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.     

Proportion of CD56+3+ T cells in decidual and peripheral lymphocytes of normal pregnancy and spontaneous abortion with and without history of recurrent abortion

PROBLEM: The present study investigated the proportion of CD56+3+ T cells in maternal peripheral and decidual lymphocytes in normal pregnancy and spontaneous abortion with and without history of recurrent spontaneous abortion (RSA). METHOD OF STUDY: Maternal peripheral blood and decidua were taken from normal pregnancies and missed abortions with and without RSA. Decidual lymphocytes were prepared from decidual tissue and analyzed by flow cytometry. RESULTS: In normal pregnancy, the percentages of CD56+3+ T cells in decidual lymphocytes did not differ from those in the peripheral blood. However, the proportion of CD56+3+ T cells in decidual CD3+ T cells increased higher than that in the peripheral CD3+ T cells. The percentages of decidual CD56+3+ T cells in missed abortions with and without RSA were lower than those in normal pregnancies. CONCLUSION: CD56+3+ T cells may play a role in the maintenance of pregnancy. The phenomenon, where the proportion of CD56+3+ T cells in decidual lymphocytes decreases, may be due to an immunologic event leading to missed abortion.     

CC chemokines induce the generation of killer cells from CD56+ cells

We describe here that members of the CC chemokines exhibit biological activities other than chemotaxis. Macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemoattractant protein-1 and RANTES, but not interleukin (IL)-8, induce the generation of cytolytic cells, designated here as CHAK (CC chemokine-activated killer) cells to distinguish them from IL-2-activated (LAK) cells. Like IL-2, CC chemokines can induce the proliferation and activation of killer cells. While incubating CC chemokines with CD4⁺ or CD8⁺ cells did not generate CHAK activity, all CC chemokines were capable of inducing CHAK activity upon incubating with CD56⁺ cells, suggesting that the primary effectors are NK cells. However, the presence of other cell types, such as CD4⁺ or CD8⁺, are necessary to induce the proliferation of CD56⁺ cells. Confirming the involvement of T cell-derived factors in inducing the proliferation of these cells, anti-IL-2 and anti-interferon-γ, but not anti-IL-1β, anti-tumor necrosis factor-α, anti-IL-8, or anti-granulocyte/monocyte-colony-stimulating factor inhibited RANTES-induced proliferation of nylon wool column-nonadherent cells. Our results may have important clinical applications for the utilization of CHAK cells in the treatment of cancer and immunodeficient patients.     

ORIGINAL ARTICLE: Women with Multiple Implantation Failures and Recurrent Pregnancy Losses have Increased Peripheral Blood T Cell Activation

Citation Yang KM, Ntrivalas E, Cho HJ, Kim NY, Beaman K, Gilman‐Sachs A, Kwak‐Kim J. Women with multiple implantation failures and recurrent pregnancy losses have increased peripheral blood T cell activation. Am J Reprod Immunol 2010 Problem We aim to determine whether peripheral blood T cell activation is associated with repeated implantation failures or recurrent pregnancy losses (RPLs). Method of study Women with a history of repeated implantation failure (n = 18) or RPLs (n = 17) comprise the study group. Normal fertile women (n = 11) are included as controls. Proportion of activated peripheral blood T cells (CD69⁺, CD154⁺) and Th1/Th2 cell ratios are measured by flow cytometric analysis. Results Proportions (%) of CD4⁺/154⁺ of CD4⁺ and CD8⁺/154⁺ of CD8⁺ cells were significantly higher in study group than those of controls. Proportions (%) of CD3⁺/69⁺ of CD3⁺ cells and CD8⁺/69⁺ of CD8⁺ cells were significantly increased in study group compared to controls. Proportion (%) of CD4⁺/69⁺ cells significantly correlated with % CD4⁺/154⁺ cells (P = 0.003). Activated cytotoxic T cells (CD8⁺/154⁺, CD8⁺/69⁺) inversely correlated with INF‐γ/IL‐10 producing CD3⁺/4⁺ T cell ratios. Proportion of activated CD3⁺/8⁺/69 and CD3⁺/8⁺/154⁺ cells was inversely correlated with IFN‐γ/IL‐10 expressing CD3⁺/4⁺ T cell ratios. Conclusion Women with MIFs or RPLs have increased T cell activation in peripheral blood lymphocytes, and T cell suppressor activation seems to be associated with decreased Th1 immunity. Further studies on T cell activation may elucidate molecular mechanisms controlling Th1 effectors.     

慢性乙型肝炎患者外周血CD8^＋ T细胞的KIR3DL1表达的探讨

目的：检测慢性乙型肝炎患者外周血CD8＋T细胞的KIR3DL1表达情况。方法：：采用流式细胞术检测慢性乙型肝炎患者外周血CD8＋T细胞的KIR3DL1分子表达,并与正常对照组比较。结果：慢性乙型肝炎患者外周血CD8＋T细胞的KIR3DL1分子表达明显高于对照组。结论：慢性乙型肝炎患者CD8＋T细胞的KIR3DL1表达显著增加。     

Expression of Inhibitory Receptors in Natural Killer (CD3CD56) Cells and CD3CD56 Cells in the Peripheral Blood Lymphocytes and Tumor Infiltrating Lymphocytes in Patients with Primary Hepatocellular Carcinoma

The cytolytic responses of NK (CD3(-)CD56(+)) and CD3(+)CD56(+) cells are inhibited by the engagement of the killer inhibitory receptors (p58.1, p58.2, and CD94) with respective ligands on the target cell. The expression of these receptors in peripheral blood lymphocytes (PBLs) (n = 18) and tumor infiltrating lymphocytes (TILs) (n = 7) was examined in patients with primary hepatocellular carcinoma (HCC). There were no differences in the expression of the three inhibitory receptors by both NK and CD3(+)CD56(+) PBLs in patients with HCC compared to that of control NK and CD3(+)CD56(+) PBLs, respectively (all P = NS). However, the expression of p58.1 by NK TILs and by CD3(+)CD56(+) TILs in patients with HCC was significantly decreased compared to that of hepatic lymphocytes of the control subjects (8.9% vs 37.85%, P = 0.047; 4.1% vs 25.2%, P = 0.049, respectively). The expression of p58.2 by CD3(+)CD56(+) TILs and CD94 by NK TILs was also decreased compared to that of hepatic lymphocytes of the control subjects (16.9% vs 73.1%, P = 0.047; 21% vs 49.95%, P = 0.037, respectively). These changes were limited to hepatic TILs, and this observation may reflect an adaptive anti-tumor phenomenon occurring in the microenvironment of HCC.     

Correlation Between Natural Cytotoxicity Receptors and Intracellular Cytokine Expression of Peripheral Blood NK Cells in Women with Recurrent Pregnancy Losses and Implantation Failures

Problem Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. We investigated whether women with recurrent pregnancy losses (RPLs) and implantation failures have aberrant correlation between NCRs and intracellular cytokine expression of NK cells. Method of study Peripheral blood NK cells (CD56^dim and CD56^bright) were analyzed for NCRs (NKp46, NKp44 and NKp30) and cytokine expression (TNF‐α, IFN‐γ, IL‐4, IL‐10) using flow cytometry in RPL (n = 22), implantation failures (n = 23) or controls (n = 15). Results In type 1 cytokine studies, CD56^bright/NKp30⁺ cells in controls (r = 0.696, P < 0.05) were positively correlated with CD56^bright/IFN‐γ⁺/TNF‐α⁺ cells. CD56^bright/NKp46⁺ cells in implantation failures (r = ?0.76, P < 0.01) were negatively correlated with CD56^bright/IFN‐γ⁺/TNF‐α^? cells. RPL did not have any correlation. In type 2 cytokine studies, CD56⁺/NKp46⁺ cells (r = 0.758, P < 0.01) and CD56⁺/NKp30⁺ cells (r = 0.637, P < 0.05) were positively correlated with CD56^bright/IL‐4⁺/IL‐10⁺ cells in controls. CD56⁺/NKp30⁺ cells in implantation failures (r = ?0.778, P < 0.05) were negatively correlated with CD56^bright/IL‐10⁺/IL‐4⁺ cells. There were no correlations in RPL. Conclusion Recurrent pregnancy losses and implantation failures have lack of, or negative correlation between NCRs and intracellular cytokines expression. This observation suggests that excessive pro‐inflammatory cytokine expression in NK cells in RPL and implantation failures may be exerted through the NCRs or interruption of signal transduction processes.     

慢性乙型肝炎患者外周血CD4＋CD25＋、CD8＋CD28＋淋巴细胞的变化及与病毒载量的关系

目的：研究慢性乙型肝炎患者外周血CD4＋CD25＋、CD8＋CD28＋淋巴细胞的表达变化及与病毒载量的关系。方法：利用流式细胞术检测50例慢性乙型肝炎患者外周血CD4＋CD25＋、CD8＋CD28＋淋巴细胞的表达率和绝对数,并与正常对照组30例进行比较;荧光定量PCR检测慢性乙型肝炎患者HBV核酸的载量,并与CD4＋CD25＋、CD8＋CD28＋淋巴细胞的表达进行相关分析。结果：慢性乙型肝炎患者的CD4＋CD25＋表达率和细胞数分别为（15.60±5.86）%和（0.34±0.13）×109/L,明显高于正常对照组（P〈0.01）;而CD4＋CD25-、CD8＋CD28＋为（21.13±5.32）%、（0.47±0.19）×109/L和（9.49±2.57）%、（0.21±0.07）×109/L,均低于正常对照组（均P〈0.01）;不同病毒载量的慢性乙型肝炎患者CD4＋CD25＋T细胞与病毒载量均呈正相关（r分别为0.552、0.588,P均〈0.01）,而CD8＋CD28＋T细胞与病毒载量均无相关性（r分别0.275、-0.092,P均〉0.05）。结论：慢性乙型肝炎患者细胞毒T细胞（CD8＋CD28＋）减少,免疫调节细胞的增加与慢性乙型肝炎患者病毒长期存在,病程迁延不愈有关。     

ORIGINAL ARTICLE: Peripheral Blood NK Cells Reflect Changes in Decidual NK Cells in Women With Recurrent Miscarriages

Citation Park DW, Lee HJ, Park CW, Hong SR, Kwak‐Kim J, Yang KM. Peripheral blood NK cells reflect changes in decidual NK cells in women with recurrent miscarriages. Am J Reprod Immunol 2010; 63: 173–180 Problem We aimed to investigate if peripheral blood natural killer (pNK) cell levels are correlated with decidual NK (dNK) cell levels, and if chemokine expression has any role in dNK cell regulation. Method of study Decidual tissues of women having two or more miscarriages with normal karyotype were collected after miscarriage and an immuno‐histochemisty study was made. pNK cells were evaluated using flow cytometric analysis. Results The %CD3^?/56⁺ and %CD3^?/56⁺/16⁺ pNK cells showed a significant correlation with mean number of CD56⁺ dNK cells. The number of decidual CD16⁺ cells was significantly higher in women with elevated pNK (≥15%) than that of normal pNK (<15%). The %CD3^?/56⁺ and %CD3^?/56⁺/16⁺ pNK cells showed an inverse correlation with duration of gestation. The CCL3⁺ and CXCL12⁺ cells were present in the decidua; however, staining intensity was not correlated with number of dNK cells. Conclusion The pNK cell levels reflect changes in dNK cell levels. This implicates that pNK cell level is a clinically useful marker to predict pregnancy outcome. Further study is needed to examine if elevated pNK cells enhance recruitment of dNK cells in the decidua.     

Down-Regulated Expression of Perforin-Positive/CD16+ Cells in the Peripheral Blood Lymphocytes in the First Trimester of Pregnancy and Up-Regulation at the End of Pregnancy

PROBLEM: Immunophenotypic profiles of perforin (P)-positive peripheral blood lymphocytes in the first trimester and at the term of human pregnancy were analyzed. METHOD OF STUDY: Perforin expression in peripheral blood lymphocyte subsets was measured by simultaneous detection of P (intracellular antigen) and cell-surface antigens (CD3, CD4, CD8, CD16, and CD56) by flow cytometry in nonpregnant (NP) and pregnant women in the first trimester (FTP) and at the time of parturition (TP). RESULTS: The percentage of total P⁺ cells in peripheral blood compared to nonpregnant women was slightly lower in the FTP but significantly higher at TP. Perforin-positive cells were significantly elevated in T lymphocyte subsets (CD3⁺P⁺, CD4⁺P\ CD8⁺P⁺) in both the FTP and TP groups, as was the percentage of CD56⁺P⁺ cells. Profound changes in the CD16⁺ subpopulation were found in the FTP group compared to both the NP and TP groups (a drastic decrease of CD16⁺P⁺ cells; CD16⁺ cells among P⁺ cells; P⁺ cells among CD16⁺ cells). A considerable part of CD3⁺ cells in both the FTP and TP groups are CD3⁺CD56⁺P⁺. The average fluorescence intensity (AFI) for P (a measure of P content per cell) was significantly decreased in FTP and increased in TP groups. CONCLUSIONS: The CD16 molecule is FcγRIIIA which is the only Fc receptor responsible for antibody dependent cell-cytotoxicity (ADCC) of NK and T-cells. In the first-trimester human pregnancy this mechanism is severely down-regulated compared to both the NP and TP groups.     

宫颈癌患者外周血CD2^＋细胞表达的检测及意义

目的：探讨宫颈癌患者外周血CD2^＋细胞表达水平及临床意义。方法：应用流式细胞术检测40例宫颈癌患者及30例宫颈上皮内瘤变（cervical intra-epithelial neoplasia,CIN）患者外周血CD2^＋细胞百分率,以30例子宫肌瘤患者作为正常对照。结果：宫颈癌患者外周血CD2^＋细胞百分率水平明显低于CIN组及正常对照组,差异均具有统计学意义（P〈0.05）。结论：CD2^＋的表达与宫颈癌的发生、发展有关,可作为宫颈癌患者治疗及评价预后的参考指标。     

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.     

A High Dose of Intravenous Immunoglobulin Increases CD94 Expression on Natural Killer Cells in Women with Recurrent Spontaneous Abortion

Problem  A high dose of intravenous immunoglobulin (HIVIg) therapy is effective in various diseases such as autoimmune diseases, and also is expected to have efficacy in recurrent spontaneous abortion (RSA). The aim of this study was to understand immunological mechanisms of this therapy.
Method of study  By flowcytometric analyses, we examined phenotypic changes of a variety of immunological cells including natural killer (NK) cells, cytotoxic T cells, regulatory T cells and macrophages in peripheral blood of RSA women with HIVIg therapy ( n  = 8).
Results  Expression percentages of inhibitory CD94 on NK cells significantly ( P =  0.01) increased after the therapy (58.8 ± 21.4% versus 71.0 ± 17.6%).
Conclusion  Mechanisms of possible efficacy of HIVIg therapy for RSA may include enhancement of CD94 expression and subsequent suppression of NK cell cytotoxicity.