Expression of NK Cell and Monocyte Receptors in Critically Ill Patients – Potential Biomarkers of Sepsis

Sepsis is characterized by activation of both the innate and adaptive immune systems as a response to infection. During sepsis, the expression of surface receptors expressed on immune competent cells, such as NKG2D and NKp30 on NK cells and TLR4 and CD14 on monocytes, is partly regulated by pro‐ and anti‐inflammatory mediators. In this observational study, we aimed to explore whether the expression of these receptors could be used as diagnostic and/or prognostic biomarkers in sepsis. Patients with severe sepsis or septic shock (n = 21) were compared with critically ill non‐septic patients (n = 15). Healthy volunteers (n = 15) served as controls. To elucidate variations over time, all patients were followed for 4 days. Cell surface expression of NKG2D, NKp30, TLR4 and CD14 and serum levels of IL‐1β, IL‐6, IFN‐γ, TNF‐α, IL‐4 and IL‐10 was estimated by flow cytometry. We found that NK cell expression of NKG2D and monocyte expression of CD14 were lower in the septic patients compared with the non‐septic patients, both at ICU admission and during the observation period (P < 0.01 for all comparisons). Both at ICU admission, and during the observation period, levels of IL‐6 and IL‐10 were higher in the septic patients compared with the non‐septic patients (P < 0.001 for all comparisons). Conclusion: As both NKG2D and CD14 levels appear to distinguish between septic and non‐septic patients, both NKG2D and CD14 may be considered potential diagnostic biomarkers of severe sepsis and septic shock.     

The use of the soluble adhesion molecules sE‐selectin,sICAM‐1, sVCAM‐1, sPECAM‐1 and their ligands CD11a and CD49d as diagnostic and prognostic biomarkers in septic and critically ill non‐septic ICU patients

Endothelial activation is pivotal in the development and escalation of sepsis. Central to endothelial activation is the endothelial up‐regulation of cellular adhesion molecules (CAMs) including E‐selectin, ICAM‐1, VCAM‐1, and PECAM‐1. Shed CAMs are also found in circulating soluble forms (sCAMs). We investigated whether sCAMs can be used as biomarkers for the differentiation between septic and non‐septic patients. Furthermore, we investigated lymphocyte and monocyte expression of LFA‐1 (CD11a/CD18) and VLA‐4 (CD49d/CD29) ligands for ICAM‐1 and VCAM‐1, respectively. Twenty‐one septic and 15 critically ill non‐septic patients were included. All patients had an APACHE II score above 13 at ICU admission. Fifteen healthy volunteers served as controls. Flow cytometry was used to estimate levels of sE‐selectin, sICAM‐1, sVCAM‐1, sPECAM‐1, and the cellular expression of CD11a and CD49d. Levels of sE‐selectin, sICAM‐1 and sPECAM‐1 were higher in the septic patients compared with the non‐septic patients and controls at admission and during the observation period. Lymphocyte and monocyte expression of CD11a and CD49d was suppressed or unaltered in the septic patients compared with the non‐septic patients and controls. Levels of sE‐selectin, sICAM‐1, and sPECAM‐1 were able to discriminate between septic and non‐septic patients, indicating that sCAMs may be potential diagnostic biomarkers of sepsis.     

Heterogeneity among septic shock patients in a set of immunoregulatory markers

Immune activation is a regular feature of sepsis, but the incidence and nature of the ensuing inflammation-resolving and immunosuppressive component is less well understood. In this study, we compared immunoregulatory markers on blood leukocytes from patients with Gram-negative or Gram-positive sepsis or septic shock, and compared this to blood from patients with severe virosis or healthy controls. To this end, blood from 32 patients with sepsis, including ten cases with shock, and 12 patients with severe virosis were analysed by flow cytometry for the expression levels of monocyte HLA-DR, CD11c, CD14 and CD40, and for frequencies of CD163⁺-suppressive monocytes, HLA-DR⁺ or CD40⁺-activated T cells and Tregs. Plasma cytokine levels were analysed as a functional measurement. Signs of immunosuppression dominated in the septic shock and Gram-positive sepsis groups, whereas monocyte activation was common in Gram-negative sepsis patients without shock. However, the main finding was the large inter-individual variation of immune activation and immunosuppression, with no correlation to prognosis among the shock patients. The pronounced inter-individual variation in the analysed monocyte and lymphocyte markers forms a strong argument that, when immunomodulatory treatment is considered in a sepsis patient, it should be personalised and guided by a detailed immune status assessment.     

Increased Expression of Programmed Cell Death‐1 in Regulatory T Cells of Patients with Severe Sepsis and Septic Shock: An Observational Clinical Study

While regulatory T cells (Tregs) constitutively express programmed cell death‐1 (PD‐1), the role of PD‐1 expression in Tregs of patients with sepsis remains unclear. Thus, we determined PD‐1 expression in Tregs from the peripheral blood of patients with severe sepsis and septic shock. Seventy‐eight patients with severe sepsis and 40 with septic shock, as well as 21 healthy subjects, were enrolled in this study. The percentage of peripheral blood PD‐1⁺ Tregs, as well as absolute Treg counts, were compared between these three groups. PD‐1 expression in Tregs and absolute Treg counts were also compared between survivors and non‐survivors in patients with sepsis. PD‐1 expression in Tregs increased in patients with sepsis compared to healthy controls. Conversely, absolute Treg counts were significantly decreased in patients with sepsis compared to healthy controls; patients with septic shock had the lowest absolute Treg counts. Among patients with sepsis, survivors had lower levels of PD‐1 expression in Tregs, as well as higher absolute Treg counts, than non‐survivors. Additionally, the percentage of PD‐1⁺ Tregs correlated positively with lactate levels as well as the Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores in patients with sepsis. PD‐1 was upregulated in Tregs of patients with sepsis and may indicate a state of immune dysfunction. Overexpression of PD‐1 in Tregs was associated with more severe sepsis as well as poor outcomes.     

The Influence of Cytomegalovirus on Expression of HLA‐G and its Ligand KIR2DL4 by Human Peripheral Blood Leucocyte Subsets

HLA‐G is a non‐classical class I HLA antigen, normally expressed in high levels only on extravillous cytotrophoblast. It has immunosuppressive properties in pregnancy and has also been found to be upregulated on leucocytes in viral infection. In this study, proportions of all leucocyte subsets expressing HLA‐G were found to be low in healthy subjects positive or negative for cytomegalovirus (CMV). Significantly greater proportions of CD4+ CD69+ and CD56+ T cells expressed HLA‐G compared to other T cells. However, following stimulation with CMV antigens or intact CMV, proportions of CD4+, CD8+, CD69+ and CD56+ T cells, and also B cells expressing HLA‐G, were significantly increased in CMV+ subjects. Despite some subjects having alleles of HLA‐G associated with high levels of expression, no relationship was found between HLA‐G genotype and expression levels. Purified B cells from CMV+ subjects stimulated in mixed culture with CMV antigens showed significantly increased HLA‐G mRNA expression by real‐time polymerase chain reaction. Serum levels of soluble HLA‐G were similar in CMV− and CMV+ subjects but levels in culture supernatants were significantly higher in cells from CMV+ than from CMV− subjects stimulated with CMV antigens. The HLA‐G ligand KIR2DL4 was mainly expressed on NK cells and CD56+ T cells with no differences between CMV+ and CMV− subjects. Following stimulation with IL‐2, an increase in the proportion of CD56+ T cells positive for KIR2DL4 was found, together with a significant decrease in CD56dimCD16+ NK cells. The results show that CMV influences HLA‐G expression in healthy subjects and may contribute to viral immune evasion.     

Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay

In present study human peripheral blood NK cell activation after co-incubation with K569 cell line was investigated by CD69 expression. NK lytic activity was studied by two different assays: TDA (2,2':6',2″-terpyridine-6,6″-dicarboxylate) release assay (TRA) and flow cytometry assay (FCA) that display two approach to cytotoxicity measurement. We also investigated NK cell degranulation activity by estimation of CD107a (LAMPa) expression. Comparison of specific lysis value measured by both cytotoxicity assays showed high correlation coefficient between two methods (r=0.94447). Specific lysis value correlated significantly with CD69+ NK frequency and NK degranulation activity. We show that lymphocyte incubation with K562 results to increase CD69 expression on NK and NKT but not on T lymphocytes. Only a part of peripheral blood NK cells became CD69 positive after incubation with excess of K562 cells. CD69+ NK cell frequencies did not increase after elevation of K562/NK ratio or incubation period that confirmed existence of subset of NK cells able to response to K562. CD69 elevation on NK significantly correlated with NK cytotoxicity (r=0.726). CD69 increases were similar when whole blood or isolated PBMC was used in assay. We also found different capacity to activation in NK subsets that express CD62L at various densities. The results demonstrated that K562 induced CD69 expression displays NK lymphocyte functional condition that associated with cytotoxic function.     

Peripheral blood monocyte and T‐lymphocyte activation levels at diagnosis predict long‐term survival in head and neck squamous cell carcinoma patients

This study was performed to determine whether peripheral blood (PB) monocyte and/or lymphocyte activation at diagnosis were associated with long‐term prognosis in patients with head and neck squamous cell carcinoma (HNSCC), and to what extent such prognostic properties relate to human papilloma virus (HPV)‐associated tumor infection of the included patients. This was a long‐term prospective study describing patient survival in relation to PB T lymphocyte and monocyte activation in patients observed for up to 14 years following diagnosis. Sixty‐four patients from a consecutive cohort of newly diagnosed HNSCC patients along with 16 non‐cancer control patients were included over a period of almost 2 years. Monocyte responsiveness was assessed at diagnosis (N = 56 HNSCC/16 non‐cancer controls) by measuring net levels of spontaneous vs lipopolysaccharide‐induced monocyte chemotactic protein (MCP)‐1 secretion in vitro. PB T lymphocyte activation was determined (N = 58 HNSCC/16 controls) by measuring the percentage of T cells expressing CD69 by flow cytometry. Whether HPV infection or not was determined by PCR analysis on formalin fixed paraffin‐embedded tumor tissue. Tumor HPV‐positive patients had better prognosis than HPV‐negative patients. A low net MCP‐1 response in monocytes predicted increased survival (Relative risk (RR) = 2.1; Confidence interval (CI): 1.1–4.0; p < 0.05). A low percentage of CD69 positive T lymphocytes also predicted better prognosis (RR = 2.6; CI: 1.3–5.0; p = 0.005). The predictive power of MCP‐1 monocyte and CD69 T lymphocyte measures were retained when adjusted for age and gender of the patients and shown to be independent of each other (N = 50 HNSCC/16 controls). The results were similar in HPV tumor‐positive and ‐negative patients. Patients with high monocyte‐ and/or T lymphocyte activation status had low survival with 8% 5 year overall survival (OS) compared to 65% 5 year OS for patients with dual low activation levels (RR = 0.27; CI: 0.14–0.56; p < 0.001), mostly secondary to disease‐specific survival. Both tumor HPV‐positive and ‐negative HNSCC patients with high percentage of CD69 positive T lymphocytes and/or high monocyte MCP‐1 secretion had low long‐term survival. The data suggest that the general inflammatory and adaptive immune systems are independently linked to the clinical aggressiveness of both tumor HPV‐negative and ‐positive HNSCC patients.     

Changes in lymphocyte subpopulations and CD3+/DR+ expression in sepsis

Objective   To detect lymphocyte subpopulations and CD3+/DR + expression in sepsis.
Methods   In a prospective clinical study we evaluated subpopulations of lymphocytes and percentage of CD3⁺/HLA-DR⁺ lymphocytes using two-color flow cytometry in 40 patients with sepsis and compared them with 34 healthy adults.
Results   Septic patients, when compared with healthy controls, have significantly lower percentage and absolute numbers of total T lymphocytes and CD4 T lymphocytes ( P  < 0.01). Absolute numbers of CD8 T lymphocytes, NK cells, CD3+/DR + lymphocytes and CD4/CD8 ratio were also decreased ( P  < 0.01). The percentage of B lymphocytes was increased ( P  < 0.01).
Conclusion   Our results are in agreement with previous findings in patients with sepsis after major surgery or trauma. The decreases in the percentage and absolute numbers of circulating lymphocyte subsets in non-surgical sepsis could represent a general reaction to stress. Increased percentage of B lymphocytes is most probably related to the bacterial etiology of the disease.     

Treg/Th17失衡在脓毒症发病机制中的作用

目的: 观察脓毒症患者外周血中CD4⁺CD2⁺CD127^-调节性T细胞(Treg)及辅助性T细胞17(Th17)的比例,并探讨Treg/Th17失衡在脓毒症发病中的作用。方法: 选择2010年1~8月入住我院ICU的脓毒症患者70例,按疾病严重程度分为脓毒症组(34例)、重症脓毒症组(21例)和脓毒症休克组(15例)。第1 d抽取外周血,应用流式细胞术检测不同组别患者外周血中Treg/Th17比例及CD14单核细胞 HLA-DR表达率,并探讨Treg/Th17比例与免疫状态及病情严重程度(APACHEⅡ评分)的关系。选取同期健康查体者为对照组(30例)。结果: (1) 和对照组相比,各病例组外周血Treg及Th17比例均明显升高(均P<0.01),但升高的程度不同。Th17表达以重症脓毒症组升高最明显,Treg表达以脓毒症休克组升高最明显。(2)Treg/Th17:脓毒症休克组>重症脓毒症组>脓毒症组,各组两两相比均有显著差异(均P<0.01)。脓毒症组Treg/Th17显著低于对照组(P<0.01),脓毒症休克组Treg/Th17显著高于对照组(P<0.01),但重症脓毒症组Treg/Th17与对照组比较无明显差异。(3)CD14单核细胞HLA-DR表达率:脓毒症休克组<重症脓毒症组<脓毒症组<对照组,两两比较均有显著差异(均P<0.01)。(4)Th17比例和APACHEⅡ评分及 HLA-DR表达率无相关性;Treg表达率和APACHEⅡ评分正相关(r=0.93, P<0.01),和HLA-DR表达率负相关(r=-0.89, P<0.01);HLA-DR表达率和APACHEⅡ评分负相关(r=-0.91, P<0.01)。结论: (1)脓毒症患者中Treg和Th17比例失调,Treg/Th17的失衡参与脓毒症的发病及进展。(1)Treg表达率可以在一定程度上反映机体病情及免疫状态。     

Somatic mosaicism caused by monoallelic reversion of a mutation in T cells of a patient with ADA-SCID and the effects of enzyme replacement therapy on the revertant phenotype

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T‐B‐ SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG‐ADA. The follow‐up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient’s clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion.     

Elevated NK Cell Cytotoxicity,CD158a Expression in NK Cells and Activated T Lymphocytes in Peripheral Blood of Women with IVF Failures

Citation Chernyshov VP, Sudoma IO, Dons’koi BV, Kostyuchyk AA, Masliy YV. Elevated NK cell cytotoxicity, CD158a expression in NK cells and activated T lymphocytes in peripheral blood of women with IVF failures. Am J Reprod Immunol 2010; 64: 58–67 Problem The aim of this study was to evaluate the role of elevated natural killer cytotoxicity (NKc) in women with multiple implantation failures (IF) in vitro fertilization–embryo transfer (IVF–ET) cycles. Methods of study Seventy‐nine antiphospholipid antibodies‐negative women with IF including 33 women with elevated NKc were selected for investigation. K‐562 cell line was used to evaluate NKc. Lymphocyte subsets, intracellular cytokines [interferon (IFN)‐γ, interleukin (IL)‐4, tumour necrosis factor, IL‐10], expression of activating markers [CD69, human leukocyte antigen (HLA)‐DR], CD8, KIR (CD158a), CD95, and chemokine receptors (CXCR3, CCR4) were estimated by flow cytometry. Results In women with IF, levels of NKc were higher than in IVF successful women. IF was associated with higher expression of CD8, CD158a, and HLA‐DR in NK cells, activating markers in T lymphocytes, and lower levels of CCR4+ and IL‐4+ T lymphocyte subsets. Predictive value of single elevated NKc for IVF success was 0.85, but addition of two other abnormal parameters resulted in its decrease to <0.39. Conclusions Elevated NKc is negative factor, though not critical for implantation in IVF cycles. Immune mechanism of IVF failure includes not only elevated NKc but also some other factors, such as elevated expression of CD8 and CD158a on NK cells, T lymphocyte activation, and diminished T helper 2 parameters.     

Changes in leucocyte and lymphocyte subsets during tuberculosis treatment; prominence of CD3dimCD56+ natural killer T cells in fast treatment responders

The immune responses against pulmonary tuberculosis are still poorly defined. This study describes changes in leucocyte and lymphocyte subsets during treatment to find reliable immunological markers for the disease and treatment response. Flow cytometric peripheral blood immune phenotyping, routine haematology and sputum microbiology were performed on 21 HIV-negative adult tuberculosis (TB) patients with positive sputum cultures during therapy in comparison with 14 healthy purified protein derivative (PPD)-positive volunteers. Patients at diagnosis showed high absolute neutrophil and monocyte counts which fell during treatment but low lymphocyte subset counts which increased [except natural killer (NK) and NK T cells]. High counts of a population of CD3(dim)/CD56+ NK T cells at diagnosis correlated significantly with negative sputum culture after 8 weeks of treatment. A multivariate classification technique showed improved correlation when NK cells were taken into account. In conclusion, peripheral blood white cell counts change significantly during treatment and counts at diagnosis, especially CD3(dim)/CD56+ NK T cells, hold promise in predictive models of TB treatment response.     

Multiple leucocyte activation markers to detect neonatal infection

Diagnosis of congenital or neonatal infection is often based on clinical signs. However, clinical symptoms of infections may not be specific, and for this reason early diagnosis is often determined on results of laboratory tests, which may not currently be adequate. A more reliable method of detection of infection may be the demonstration of activated lymphocytes, which can be conducted rapidly and before the isolation of the infected organism. We have shown that detection of up-regulation of CD45RO, an activated/memory isoform of CD45 present on T cells, provides a reasonably sensitive screening test for neonatal infection. We also showed that dual expression of CD45RA/CD45RO was up-regulated early during the infective process in neonates with documented infection. However, other leucocytes are also activated during the infective process. To improve the sensitivity of the neonatal infection screening test and to identify the types of leucocytes involved in the immune response to the infective organism, we studied further the up-regulation of a comprehensive range of surface activation markers on T cells, monocytes and natural killer (NK) cells from a group of 17 newborn patients with positive culture, a group of 40 possibly infected patients based on clinical signs and a control group. 'Normal' ranges were established for each activation marker for each leucocyte subset from 1 to 7 and 7-14-day-old newborns <35 weeks' gestation and 35-40 weeks' gestation. There was a significant increase in the percentage of T cells expressing CD25 in the peripheral blood from infants at 2 weeks of age. Expression of HLA-DR on T cells, CD25 and CD69 on monocytes and HLA-DR on NK cells was also increased significantly in the peripheral blood from infants at 2 weeks of age and may reflect a maturation of these functional surface molecules. Up-regulation of CD69 on NK cells was the most sensitive marker for neonatal sepsis (positive in 13/16 patients). CD69 and CD25 expression was increased significantly on T cells in 11/17 and 10/17 patients, respectively. A combination of CD45RA/CD45RO and CD45RO identified 11/16 infected patients. Measurement of CD69 expression on NK cells with CD45RA, CD45RO, CD25 and CD69 expression on T cells resulted in a significant increase in at least two leucocyte activation markers from infected patients. In conclusion, this is the first report of the up-regulation of CD69 on NK cells as a sensitive marker of neonatal infection. A combination of this marker with CD45RA, CD45RO, CD25 and CD69 expression on peripheral blood derived T cells is the most sensitive and specific for neonatal infection.     

非结核分枝杆菌感染患者外周血免疫细胞改变及其临床意义

目的分析非结核分枝杆菌(nontuberculosis mycobacterium,NTM)感染患者外周血免疫细胞表达率的改变以及临床意义.方法选择2016年4月至2017年4月期间苏州市第五人民医院住院的69例NTM感染患者作为病例组,选取同期本院健康体检者66例作为对照组.采用流式细胞术对健康对照组和NTM感染患者(NTM组)外周血免疫细胞表达率进行检测,并比较各组之间的差异.进一步根据感染部位的差异将NTM组分为单侧感染组(29例)和双侧感染组(40例),比较两组之间免疫细胞表达水平的差异.结果与对照组相比较,NTM组的总的T淋巴细胞表达率没有显著改变,而B淋巴细胞与单核细胞表达率出现显著上调[(10.50±0.56)%比(12.26±0.57)%,(5.61±0.24)%比(6.72±0.32)%,t值分别为2.209和2.774,P值均<0.05],自然杀伤(natural killer,NK)细胞表达率出现显著下调(21.87±0.98)%比(18.63±0.98)%,t=2.341,P<0.05).对T细胞亚群的分析发现,NTM组的CD4+T细胞、CD8+T细胞、CD8+CD28-T细胞的表达率与对照组相比,差异无统计学意义(P>0.05),而CD4+CD25HT细胞的表达率较对照组显著上调[(8.05±0.35)%比(3.69±0.19)%,t=11.000,P<0.05],CD8+CD28+T细胞表达率较对照组显著下调[(23.76±0.90)%比(27.07±0.74)%,t=3.039,P<0.05].进一步依据患者感染部位比较分析发现,单侧感染组与双侧感染组的T淋巴细胞、B淋巴细胞、NK细胞、单核细胞在外周血中表达率的差异均无统计学意义(P>0.05).T细胞亚群的结果分析显示,CD4+T细胞、CD8+T细胞、CD4+CD25HT细胞在单侧感染组与双侧感染组之间的表达率差异无统计学意义(P>0.05),而双侧感染组的CD8+CD28+T细胞表达比例较单侧感染组显著下降[(21.99±1.02)%比(26.21±1.52)%,t=2.397,P<0.05]、CD8+CD28-T细胞表达比率较单侧感染组显著上调[(24.48±1.90)%比(18.19±1.60)%,t=2.404,P<0.05].结论NTM感染患者外周血中一些免疫细胞的表达率出现了显著改变,可能对疾病的发生与发展产生了重要影响.     

Downregulation of CD40 ligand response in monocytes from sepsis patients

It has been suggested that a defective adaptive immune response contributes to septic immunosuppression. Here, the response of monocytes to CD40 ligand (CD40L) for patients with sepsis due to infection with gram-negative organisms has been analyzed. Compared to cells from controls, monocytes from septic patients showed significantly reduced production of tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-12 and were unable to acquire high levels of CD80 and CD86 molecules. These alterations were observed at the onset of sepsis and persisted at day 7. However, the ability of monocytes to respond to CD40L stimulation was partially but significantly restored in cells from patients who recovered from sepsis. In addition, costimulation of autologous CD4⁺ T lymphocytes by CD40L-activated monocytes from septic patients failed to induce cell proliferation and gamma interferon production. Finally, the ability of CD40L to rescue monocytes from apoptosis was severely impaired. We conclude that downregulation of the CD40L response may be an appropriate model for the monocyte alteration observed during septic immunosuppression and may help in the development of novel therapeutic strategies.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus , bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4⁺ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25⁺ and CD69⁺ lymphocytes were higher. Seventy-four percent of the CD25⁺ PBMC but only 55% of the CD69⁺ cells were CD3⁺ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69⁺ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

新辅助化疗对局部进展期乳腺癌患者T淋巴细胞亚群及NK细胞免疫功能的影响

目的:探讨局部进展期乳腺癌患者新辅助化疗前、后T淋巴细胞亚群及NK细胞免疫功能的变化。方法:采用流式细胞术检测54例局部进展期乳腺癌患者新辅助化疗前后的静脉血T淋巴细胞亚群及NK细胞免疫功能。美国癌症联合会(American Joint Commitree on Cancer,AJCC)肿瘤分期为Ⅱb期(仅T3N0M0)和Ⅲ期(不包括N3),静脉血于第1周期新辅助化疗治疗前及第3周期化疗后21日抽取,淋巴细胞亚群检测包括:T(CD3+,CD4+,CD8+),NK(CD56+,CD16+),经过3周期新辅助化疗CEF方案(表柔比星、环磷酰胺和5-氟尿嘧啶),根据新辅助化疗临床效果评价分为2组,化疗有效组38例(CR和PR),化疗无效组16例(SD和PD),并与正常体检健康者(40例)作比较。结果:乳腺癌患者治疗前CD4+、CD4+/CD8+明显低于对照组(P<0.01),NK细胞明显低于对照组(P<0.05),新辅助化疗后,有效组总CD3+、CD4+、CD4+/CD8+、NK细胞较治疗前均显著升高(P<0.05),CD8+降低(P<0.05);无效组CD3+、CD4+/CD8+及NK细胞较治疗前显著降低(P<0.05),而CD8+升高(P<0.05)。结论:局部进展期乳腺癌患者免疫功能低下,有效的辅助化疗能提高患者的免疫功能,定期监测免疫功能对指导临床治疗有意义。     

NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease

During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.     

外周血CD3~+CD56~+T细胞在恶性肿瘤患者中的表现及临床意义

目的了解 CD3+ CD5 6 + T细胞与 CD3- CD5 6 + 、CD3+ CD5 6 - 的关系及其在参与恶性肿瘤患者抗肿瘤免疫中的作用。方法采用流式细胞术对 10 0例恶性肿瘤患者 (5 5例实体瘤患者和 45例非实体瘤患者 )及 46例健康对照组外周血中的 CD3+ CD5 6 +、CD3- CD5 6 +、CD3+ CD5 6 - 3类淋巴细胞进行标记分析。结果在实体瘤和非实体瘤患者组中 :CD3+ CD5 6 + T细胞均有高表达 ,2组患者与健康对照组比较均有显著性差异 (P<0 .0 1)。 CD3+ CD5 6 - T细胞在实体瘤组的表达都显著低于非实体瘤组和健康对照组 ,2组间比较均有显著性差异 (P<0 .0 0 1) ;而 CD3- CD5 6 + NK细胞在 2患者组中的表达与健康对照组比较均无显著性差异 (P>0 .0 5 )。结论 CD3+ CD5 6 + T细胞在恶性肿瘤患者外周血中的高表达较 CD3- CD5 6 + NK细胞更明显 ,并且不受恶性肿瘤细胞类型的影响 ,提示高表达的 CD3+ CD5 6 + T细胞是参与抗肿瘤免疫的重要表现     

Expression profiles of peripheral CD160+ lymphocytes during the course of healthy human pregnancy

Citation
Barakonyi A, Weisdorn R, Miko E, Varga P, Bodis J, Szekeres‐Bartho J, Szereday L. Expression profiles of peripheral CD160⁺ lymphocytes during the course of healthy human pregnancy. Am J Reprod Immunol 2011; 66: 137–142 Problem CD160 receptor is expressed by natural killer (NK) and T‐cell subsets, and after activation, it could enhance cytotoxicity or pro‐inflammatory cytokine production on NK cells. Here, we investigated the phenotype of peripheral CD160⁺ cells during healthy pregnancy. Method of study We analyzed the expression of CD69 activation marker, gamma/delta TCR, and NKG2A or NKG2D NK cell receptors on CD160⁺ lymphocytes of non‐pregnant and healthy pregnant women at four different stages of pregnancy by flow cytometry. Results In our hands, CD160 receptor‐positive lymphocytes were present during pregnancy; however, they had different characteristics depending on gestational age. During implantation, CD160⁺ cells showed low activation rate, decreased NK receptor expression while 40% of Vδ2 + T cells expressed CD160 receptor. In turn, all the above parameters increased as pregnancy proceeds. Conclusion Our results indicate that CD160⁺ lymphocytes could be able to play a role in the maintenance of healthy pregnancy.