Percentage Of CD4 CD8 Research Articles

Clinical value of CD3-CD56+ natural killer cells, IL-2, and IL-8 in acute exacerbation of chronic obstructive pulmonary disease in patients with respiratory failure.

This study aims to investigate the the content of CD3-CD56+ Natural Killer (NK) cells in peripheral blood and the serum interleukin-2 (IL-2) and interleukin-8 (IL-8) levels in patients with acute exacerbation of Chronic Obstructive Pulmonary Disease (AECOPD) complicated by respiratory failure (RF). Besides, their diagnostic and prognostic values for AECOPD combined with RF were also explored. This retrospective study included patients from the Affiliated Jiangning Hospital of Nanjing Medical University between December 2021 and December 2023. A total of 65 AECOPD patients with RF were selected as the RF group, 64 AECOPD patients without RF as the AE group, and 60 patients with stable COPD as the COPD group. Data on gender, age, course of disease, smoking, alcohol consumption, history of hypertension and diabetes were collected. Serum levels of IL-2 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Arterial oxygen partial pressure (PaO2) and arterial carbon dioxide partial pressure (PaCO2) at admission were measured by automatic blood gas analyzer. Pulmonary function was assessed using forced expiratory volume in the first second (FEV1)/forced vital capacity (FVC) and the percentage of FEV1 to the predicted value (FEV1%). The percentage of NK cells (CD3-CD56+) was detected by flow cytometry. After discharge, patients were followed for one year and categorized into a favorable prognosis (FP) group or an unfavorable prognosis group (UP) based on the severity of lung function impairment. Expression levels of IL-2 in the COPD group, AE group, and RF group were 2.91±0.55, 2.21±0.48 and 1.39±0.43, respectively, while the expression levels of IL-8 were 31.01±4.86, 38.02±5.16 and 44.43±6.54, respectively. The percentages of CD3-CD56+ NK cells in the three groups were 19.93±2.40%, 22.57±3.70% and 25.48±3.64%, and the levels of FEV1/FVC were 60.81±6.00%, 51.31±4.95% and 42.67±8.77%, respectively. FEV1% were 61.11±5.71%, 45.45±6.86% and 38.77±10.07%, and PaCO2 levels were 44.08±4.91 mmHg, 53.02±10.52 mmHg and 65±6.63 mmHg, respectively. PaO2 levels were 81.5±5.64 mmHg, 59.1±5.95 mmHg and 53.86±7.06 mmHg, respectively. The differences in the above indices were statistically significant among the three groups (all P<0.05). In all COPD patients, IL-2, IL-8, and CD3-CD56+ NK cells (%) were associated with lung function and arterial blood gas values. In addition, these markers demonstrated predictive value for the prognosis of AECOPD patients complicated by RF, with their combined detection showing a higher predictive value. The combined assessment of serum IL-2, and IL-8 levels and positive CD3-CD56+ NK cell rate in peripheral blood of AECOPD patients has diagnostic value for identifying AECOPD with RF and predicting poor prognosis. These markers can serve as predictors of outcomes.

The Effect of Acute Physical Exercise on Natural Killer Cells Populations and Cytokine Levels in Healthy Women.

Physical exercise generates a systemic response in the immune system. It has been observed that cell populations respond to exercise stimuli, especially Natural Killer cells, whose number increase within minutes of starting physical exertion. This study aimed to evaluate the acute effect of moderate- and high-intensity exercise on immunological markers in healthy women. As specific objectives, the percentages of CD3-CD56+ Natural Killer total cells, CD56brightCD16dim effector subpopulation, CD56dimCD16bright cytotoxic subpopulation, NKG2A inhibition receptor, NKG2D activation receptor, and NKT cells were analyzed. In addition, the levels of the cytokines IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF and the chemokines CCL5/RANTES, CXCL9/MIG, CCL2/MCP-1, and CXCL10/IP-10 were also analyzed. Natural Killer total cells showed an increase in their percentage in both exercise protocols (p = 0.001 for the moderate-intensity group and p = 0.023 for the high-intensity group); however, only in the high-intensity exercise session was there an increase in the CD56dimCD16bright cytotoxic subpopulation (p = 0.014), as well as a decrease in CD56brightCD16dim effector subpopulation (p = 0.001) and their NKG2A inhibition receptor (p = 0.043). An increase in IL-6 was observed after the high-intensity exercise session (p = 0.025). Conclusions. Physical exercise influences immunological markers and shows an acute response to moderate- or high-intensity exercise.

The predictive value of NKG2C+NK cells and LILRB1+NK cells in recurrent spontaneous abortion.

The maternal-fetal immune abnormalities can result in recurrent spontaneous abortion (RSA). The role of NKG2C+ and LILRB1+ pNK subsets in predicting pregnancy loss is uncertain. In this study, we aimed to compare the percentage of CD3- CD56+ NK cells, NKG2C+ NK cells, and LILRB1+ NK cells in peripheral blood between healthy pregnant women (HC group), RSA women followed by normal pregnancy (RSA-N group) and RSA women followed by abortion (RSA-A group) in the first trimester via flow cytometry, and explore the prediction value of NKG2C+ and LILRB1+ NK cells for pregnancy loss in RSA via ROC curve. The MFI of NKG2C and LILRB1 of dNK were compared between and HC and RSA-A groups. The percentage of CD3-CD56+ pNK cells between HC, RSA-N, and RSA-A groups shows no significant difference. In peripheral blood, the percentage of NKG2C+ NK cells were significantly increased in the RSA-A group than HC group and RSA-N group, and the percentage of LILRB1+ NK cell were significantly decreased in the RSA-A group. The MFI of NKG2C and LILRB1 of dNK showed a similar trend with peripheral blood between HC and RSA-A groups. The NKG2C+ and LILRB1+ NK cells were an independent risk factor for predicting pregnancy loss in RSA patients, with an area under the ROC curves (AUC) of .77 and .71 respectively. Women with recurrent spontaneous abortion have abnormal NKG2C+ and LILRB1+ pNK subsets, which could reflect immune abnormalities at the maternal-fetal interface, and NKG2C+ and LILRB1+ pNK subsets could be a good indicator for the prediction of pregnancy loss.

Defective expansion and function of memory like natural killer cells in HIV+ individuals with latent tuberculosis infection.

PurposeTuberculosis (TB) is the leading cause of infectious disease related mortality, and only 10% of the infected individuals develop active disease. The likelihood of progression of latent tuberculosis infection (LTBI) to active TB disease is high in HIV infected individuals. Identification of HIV+ individuals at risk would allow treating targeted population, facilitating completion of therapy for LTBI and prevention of TB development. NK cells have an important role in T cell independent immunity against TB, but the exact role of NK cell subsets in LTBI and HIV is not well characterized.MethodsIn this study, we compared the expansion and function of memory like NK cells from HIV-LTBI+ individuals and treatment naïve HIV+LTBI+ patients in response to Mtb antigens ESAT-6 and CFP-10.ResultsIn freshly isolated PBMCs, percentages of CD3-CD56+ NK cells were similar in HIV+LTBI+ patients and healthy HIV-LTBI+ individuals. However, percentages of CD3-CD56+CD16+ NK cells were higher in healthy HIV-LTBI+ individuals compared to HIV+LTBI+ patients. HIV infection also inhibited the expansion of memory like NK cells, production of IL-32α, IL-15 and IFN-γ in response to Mtb antigens in LTBI+ individuals.ConclusionWe studied phenotypic, functional subsets and activation of memory like-NK cells during HIV infection and LTBI. We observed that HIV+LTBI+ patients demonstrated suboptimal NK cell and monocyte interactions in response to Mtb, leading to reduced IL-15, IFN-γ and granzyme B and increased CCL5 production. Our study highlights the effect of HIV and LTBI on modulation of NK cell activity to understand their role in development of interventions to prevent progression to TB in high risk individuals.

HYPOXIA-HINDERED METHYLATION OF PTGIS IN ENDOMETRIAL STROMA CELLS ACCELERATES ENDOMETRIOSIS PROGRESSION BY INDUCING CD16- NK CELLS DIFFERENTIATION

To investigate the role of prostacyclin (PGI2) in the pathogenesis of endometriosis (EMS). Expression of prostacyclin synthase (PTGIS)/PGI2 in normal (nEM) and ectopic endometrium (eEM), normal (NESCs) and ovarian endometrial stroma cells (OESCs) were analyzed in Gene Expression Omnibus (GEO) and further verified with quantitative Real-Time PCR (qRT-PCR), immunohistochemistry (IHC), western blotting (WB), enzyme-linked immuno sorbent assay (ELISA) and flow cytometer (FCM) assays. Methylation status of PTGIS promoter was detected in NESCs, OESCs and hypoxia-conditioned human endometrial stroma cells (HESCs). PTGIS over-expressed HESCs underwent RNA-sequencing process and co-cultured with the human peripheral natural killer (pNK) cells, and percentage of CD3-CD56+CD16- (CD16-) pNK cells were detected. Additionally, role of PTGIS/PGI2 in EMS was explored in vivo by instructing PTGIS knocked out (KO) (PTGIS-/-) mice, CD16 KO (CD16-/-) mice and mouse spleen NK cells adoptive transferring EMS model. Lastly, statistical analysis was analyzed by Student's t-test, one-way analysis of variance (ANOVA) and Mann-Whitney test. The expression of PTGIS/PGI2 was augmented in EMS specimens, which attributed to hypoxia milieu in endometriotic lesions and hypoxia-inducible factor 1 alpha (HIF-1α) accelerated PTGIS transcription through DNMT1-mediated DNA methylation deficiency in PTGIS promoter. Furthermore, over-expression of PTGIS enhanced adhesion ability of ESCs and ESCs derived PGI2 promoted CD16- NK cells differentiation through PGI2 receptor (PTGIR) in vitro. More importantly, PGI2 analogue iloprost and CD16- mouse spleen NK cells adoptive transfer aggravated EMS severity, with PTGIS-/- uterus fragments recipient and PTGIR antagonist RO1138452 treatment EMS model being endowed with attenuated ectopic lesions in vivo. Endometriotic hypoxia milieu activated PTGIS/PGI2 signaling pathway through DNMT1-mediated DNA methylation depression in PTGIS promoter, and this pathway accelerated EMS progression by enhancing adhesion ability of ESCs and inducing CD16- NK cells differentiation.

Both CD4+CD25+Foxp3+ Regulatory T Cells and CD3-CD56+ NK Cells Affect the Response of Newly Diagnosed Chronic Myeloid Leukemia Patients to Treatment with a Tyrosine Kinase Inhibitor

Background: Regulatory T cells (Treg) play a role in the maintenance of chronic myeloid leukemia (CML). In contrast, natural killer (NK) cells have spontaneous cytolytic activity against various tumor cells, including leukemic cells, and play an important role in immune surveillance against tumor cells. In this study, we examined changes in the number of circulating Treg and NK cells and the NK activity of patients with CML before and after treatment with a tyrosine kinase inhibitor (TKI). Our goal was to evaluate the clinical significance of this treatment on these two different cell types.Patients and Methods: Treg and NK cells were characterized and quantified by flow cytometry in 13 newly diagnosed CML patients, 5 newly diagnosed patients with other myeloproliferative neoplasms (MPN), and 15 healthy controls. We analyzed all CML patients diagnosed with chronic phase (CP) disease and MPN patients from January 2015 and June 2016. Patients with CML, who were initially treated with TKI between January 2015 and June 2016 and were followed up for more than one year, were enrolled in the study. The mRNA level of the BCR-ABL fusion gene was analyzed by real-time PCR analysis at approximately 6-month intervals during the first year and at 12-month intervals thereafter. After obtaining informed consent, blood was drawn from all patients at initial diagnosis and 12 months after the start of treatment. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at effector: target (E: T) ratios of 20:1.Results: The median age for CML patients was 65 years (range: 47-84), and 9 of the patients were male. The median period of observation was 543 days. After 12 months, 8 CML patients (MMR-group) had a major molecular response (MMR), and 5 patients (NR-group) did have not a MMR. Before treatment, the absolute number of Treg cells was significantly lower in the MMR-group than that in the NR-group (27.6 ± 10 vs. 50.0 ± 24.0 /μL, p=0.037). Similarly, the absolute number of Treg cells after treatment was significantly lower in both the MMR-group (27.3±10.3 /μL) and the MPN-group (27.6±11.5 /μL) than that in the NR-group (40.0±9.9 /μL, p=0.05, p<0.01, respectively). Treg% values in peripheral lymphocytes before treatment in the NR (7.4±2.1%) were significantly higher than that in MMR group and in the healthy group (4.7±1.7%, 5.5±1.1%, p=0.024, p=0.016, respectively). The absolute number and percentages of CD3-CD56+ NK cells before treatment as well as after treatment did not differ between each group. However, NK activity before treatment in the NR-group (15±1.4%) was significantly lower than that in MMR-group (47.2± 11.6 vs 41.7±4.1%, p<0.01, p<0.01, respectively). With time, the number of CD8+ T cells in the NR group decreased significantly from 940±570 to 348± 167/μL (p=0.040), and the Treg% increased significantly from 0.18± 0.13 to 0.84± 0.29% (p<0.01). The relative percentage of CD3+CD4+ T cells (4.7±4.4%) in the 15 CML patients increased remarkably after treatment (12.8±4.3%, p<0.01) but the absolute number did not change.Conclusions: Treg cell numbers were significantly lower in the CML patients with a MMR compared to that in the patients without a MMR. Although the number of NK cells did not change in the MMR group compared to that in the NR group, the NK activity was significantly higher. While the mechanisms for these changes in cell populations are not completely understood, it is also important to note that we have not yet studied the cellular source and production of cytokines which enhance NK activity. The data suggest that some immunological mechanisms are involved in the response to TKI. Moreover, the increased levels of NK activity in patients with MMR suggest that not only Treg cell numbers but also NK activity may have clinical importance in evaluating the effects of TKI therapy. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Bone Marrow Fibrosis and Lymphocyte Subsets in Adult Patients with Primary Immune Thrombocytopenia and Correlation with Clinical Findings

Immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by low platelet counts that can lead to bleeding. Diagnosis of ITP is generally made by review of peripheral smear and evaluation of history and examination of the patient. Bone marrow examination is not diagnostic in patients with ITP and is performed only in those with other hematologic abnormalities and in those who do not have an adequate response to treatment.Bone marrow examinations in ITP have been investigated in studies dating back several decades. Some investigators have found megakaryocyte numbers to be increased . Meanwhile,other study have shown reticulin levels among adult patients with primary ITP may be elevated with respect to the general adult population. Morphological changes in ITP MKs include cytoplasmic vacuolization, reduction of granules, and smoothing of plasma membrane associated with increased number of MKs in BM. However, fibrosis appears to be no correlation with disease progression and the correlation with clinical outcome remains equivocal at best.The current study aimed to understand the marrow levels of reticulin and/or collagen fibres among adult patients-the majority with clinical features of primary ITP-at the time of diagnosis and to evaluate any correlation between fibrosis and primary ITP specific clinical features. Results 1. Of the 282 newly diagnosedand chronic patients from October 2015 to November 2017 were enrolled into the study which 102 patients were newly diagnosed and 182 patients were chronic.Median age was 46 yr (range 18-85), with 108 males and 174 females. At diagnosis 229 (79%) patients were age 60 or younger and 62 (81.8%) were women. There were more females than males with no significant difference in age between genders. CD41+megakaryocyte numbers on bone marrow aspirates did not meet statistical significance, a trend that chronic group likely have more megakaryocyte numbers189(IQR 68-340) than newly diagnosed group143(IQR 70-309). The prevalence of a bone marrow grading greater than 0 among our sample of newly diagnosed and chronic ITP patients were 23.5% and 14.4%, but the difference was not statistically significant(P=0.063). 2. Comparing cases with BMF&amp;gt; 0 with those without (MF=0),the former showed increased bone marrow cellularity (P=0.022).In terms of changes in lymphocyte subsets, the percentage of CD19+B Lymphocyte and CD3-CD56+ NKLymphocyte in patients with bone marrow grading &amp;gt; 0 were obviously lower than those in bone marrow grading = 0[8 (4.1-12.4) vs 5.0 (3.6-8.8), P=0.008].The bone marrow fibrosis grade were inversely correlated with the percentage of CD3-CD56+ NK Lymphocyte(r=0.307, P=0.004). In a further analysis, a significant inverse correlation was present between the CD3-CD56+ NK Lymphocyte and bone marrow cellularity(r=0.432, P=0.008). 3. In newly diagnosed patients who had achieved R, the median (range) TTR was 5 days (6-14.5 days) in the bone marrow grading =0 group compared with 6 days (6-18 days) in bone marrow grading &amp;gt; 0 group (P =0.624). Notably, in chronic ITP patients ,MF&amp;gt; 0 cases more frequently required more therapies compared with bone marrow grading = 0 group 2 (range, 1-4) vs 4((range, 4-5) during the first year after diagnosis (P&amp;lt;0.01). twenty-seven (37.5%) of the newly diagnosed 72 patients were long-term responders. 4. Factors affecting response were assessed by univariate logistic regression, which indicated that CD3-CD56+NK Lymphocyte was independently associated with a higher response (P = 0.010; hazard ratio (HR)0.515, 95% CI 0.311-0.855). As for the factors affecting LTR, univariate logistic regression results in Table 4 showed that MF&amp;gt; 0 , CD19+B Lymphocyte and gender is a predictive factor for achieving LTR(0.009; hazard ratio (HR)0.789 , 95% CI 0.561-0.762). As a conclusion, BM fibrosis is not rare in ITP patients,its incidence is actually higher than expected, as stated in literature. In this study, there were differences between ITP-associated MF and without MF in terms of number of re-treatment ,LTR, lymphocyte subsets in ITP. However, the TTR were no differences between ITP-associated MF and without MF patients. Disclosures No relevant conflicts of interest to declare.

Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway.

Acute myeloid leukemia (AML) is a common type of hematological malignancy that can progress rapidly. AML has a poor prognosis and a high incidence of relapse due to therapeutic resistance. Azelaic acid (AZA), a small molecular compound is known to exhibit antitumor effect on various tumor cells. This study aimed to evaluate the antiproliferative and immunoregulatory effects of AZA against AMLviathe activation of the notch signaling pathway. We found that AZA can inhibit the proliferation of AML cells. In addition, laser confocal microscopy showed AZA-treated AML cells began to swelling and undergo cytoplasmic vacuolization. Importantly, AZA promoted the proliferation of NK and T cells and increased the secretion of TNF-αand IFN-γ. AZA also increased the expression levels of CD107a and TRAIL in NK cells, and CD25 and CD69 in T cells to influence their activation and cytotoxic ability. AZA-treated NK cells can kill AML cells more efficiently at the single-cell level as observed under the microfluidic chips. Further mechanistic analysis using protein mass spectrometry analysis and Notch signaling reporter assay demonstrated that Notch1and Notch2 were up-regulated and the Notch signaling pathway was activated. Moreover, combining AZA with the Notch inhibitor, RO4929097, decreased the expression of Notch1and Notch2, and downstream HES1 and HEY1, which rendered AML cells insensitive to AZA-induced apoptosis and alleviated AZA-mediated cytotoxicity in AML. In vivo, AZA relieved the leukemic spleen infiltration and extended the survival. The percentage of CD3-CD56+NK cells and CD4+CD8+T cells as well as the secretion of cytotoxic cytokines was increased after the treatment of AZA. The overall findings reveal that AZA is a potential Notch agonist against AML in activating the Notch signaling pathway.

Abstract LB-072: Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2

Abstract Introduction: Generating clinical scale doses of natural killer (NK) cells is currently a challenge for manufacturing CAR-NK cell therapies. Current methods to expand NK cells include irradiated feeder cells that have been engineered to mimic dendritic cells as well as commercially available combinations of antibody &amp; proteins for targeted NK cell expansion. These methods are restrictive due to high costs, scale-up challenges, and licensing restrictions for clinical &amp; commercial applications. We hypothesized that using a dissolvable phase-change hydrogel conjugated with ligands targeting NK cell activation may provide a more efficient and defined alternative for NK manufacturing processes. Additionally, we investigated cytokine culture combinations that optimize the expansion of NK cells. Methods: Hydrogel microspheres (9 µm median diameter) - referred to as Cloudz™ particles - were functionalized with αNKp46 and αCD2 ligands using standard bioconjugation techniques. In this study, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in G-Rex® 24-well plates containing NK cell-functionalized Cloudz™ particles along with various cytokine combinations. Prior to expansion, PBMCs were analyzed for CD3-/CD56+ NK cells and 6x105 NK cells were seeded into each well with a 2:1 ratio of Cloudz™ particles:NK cells. Miltenyi MACS iBeads were used as a control and were prepared according to the manufacturer instructions. During days 0-3, the cells were cultured with IL-2 (40 U/mL) along with combinations of IL-12, IL-18, and IL-21 (10 ng/mL each). After 3 days, the IL-2 concentration was increased to 200 U/mL, while the other cytokine concentrations remained constant. The cytokines and media were refreshed every 3 days with ½ media changes. After 7 days in culture the cells were analyzed by flow cytometry for CD3-/CD56+ NK cell expansion and purity. NK cell purity was defined as the percentage of CD3-CD56+ cells relative to the total population. Results: Cultures using NK cell-functionalized Cloudz™ particles combined with IL-2 resulted in a 69% NK cell purity and 29.9-fold expansion. In comparison, cultures using MACS iBeads combined with IL-2 resulted in 49% NK cell purity and a 6.8-fold expansion, suggesting that Cloudz™-based methods provide better expansion. When IL-12 was added to cultures with Cloudz™ particles, both the purity and expansion decreased (45% and 7.2-fold, respectively). When IL-12 and IL-18 were added, the expansion increased to 33.9-fold and the purity increased to 60%. The best combination was IL-2/IL-12/IL-18/IL-21 which had an 88.9-fold expansion with a 78% purity. IL-12 and IL-18 mimic the activating signals released by dendritic cells and IL-2 and IL-21 mimic the activating signals released by activated T cells. Conclusion: These data demonstrate that Cloudz™ particles functionalized with αNKp46 and αCD2 can efficiently induce NK activation and expansion with high purity and growth. In future studies, we will determine if NK cells expanded using this method show killing ability of cancer cells and persistence in in vivo models. We will also further explore the possibilities of this novel system for rapid expansion and recovery of specific NK cell subtypes. Citation Format: Christopher Johnson, Hannah Senior, Nithya Jesuraj. Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-072.

Proportion of Cytotoxic Peripheral Blood Natural Killer Cells and T-Cell Large Granular Lymphocytes in Recurrent Miscarriage and Repeated Implantation Failure: Case-Control Study and Meta-analysis.

We aimed to compare the proportion of peripheral blood natural killer (NK) cells (CD3-CD56+) and T-cell large granular lymphocytes (CD8+CD57+) during preconception in a homogenous group of women with unexplained well-defined recurrent miscarriage (RM) and repeated implantation failure (RIF) vs healthy controls in relation to pregnancy outcomes. This case-control study followed by a literature review and meta-analysis was conducted in three university hospitals. Patients and controls were consecutively recruited from December 2015 to October 2017. In total, 115 women were included in the study: 54 with RM, 41 with RIF and 20 healthy controls with ≥ 2 term births. Percentages of CD3-CD56+ and CD8+CD57+ cells and sub-populations of CD3-CD56+ cells did not differ between cases and controls. The results for women with subsequent miscarriage did not differ from those with live births. The meta-analysis of the literature showed higher NK-cell proportions in RM [mean difference 3.47 (95% CI 2.94-4.00); p < 0.001] and RIF [mean difference 1.64 (95% CI 0.82-2.45); p < 0.001] than controls. However, the heterogeneity between the different studies was high. The proportion of peripheral blood CD3-CD56+ and CD8+CD57+ cells in the preconception period does not reflect the risk of implantation failure or miscarriage and should not be recommended indicators for the management of RM and RIF. Further prospective large studies are needed to develop a reliable peripheral blood marker of immune deregulation.

Changes in Both CD4+CD25+Foxp3+ Regulatory T Cells and CD3-CD56+ NK Cells in Patients with Chronic Myeloid Leukemia Treated with a Tyrosine Kinase Inhibitor

Background: The immunological graft-versus-leukemia effects associated with chronic myeloid leukemia (CML) are well-established. Both regulatory T cells (Treg) and natural killer (NK) cells play a role in the maintenance of CML. In this study, we examined changes in the number of circulating Treg and NK cells and the NK activity in CML patients before and after treatment with a tyrosine kinase inhibitor (TKI). Our goal was to evaluate the clinical significance of this treatment on these two different cell types. Moreover, the levels of these cells suggest that there is a need to make the TKI more effective as an immunotherapy.Patients and Methods: Treg and NK cells were characterized and quantified by flow cytometry in 27 newly diagnosed CML patients, 19 newly diagnosed patients with other myeloproliferative neoplasms (MPN), and 15 healthy controls (HC-group). CML patients, who were diagnosed as chronic phase and initially treated with a TKI between September 2007 and July 2017 and were followed up for more than 12 months were enrolled in the study. The molecular response was analyzed by quantitative PCR of the BCR-ABL fusion genes at approximately 6-month intervals after the initiation of treatment. After obtaining informed consent, peripheral blood (PB) was drawn from all patients at their initial diagnosis and at 6-month intervals thereafter. CD4+CD25+Foxp3+ Treg cells, CD3+CD4+T cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry, and the absolute numbers of these cells were calculated. The NK activity was measured by the standard 51Cr release assay at an effector:target (E:T) ratio of 20:1.Results: The median age for CML patients was 65 years (range: 44-85), and 17 out of 27 of the patients were male. The median period of observation was 656 days. After 12 months, 12 CML patients (MMR-group) had a major molecular response (MMR), and 15 patients (NR-group) did not have a MMR. Treg% values in the PB before treatment in the CML-group (0.39±0.30%) were significantly lower than that in the HC-group (0.70±0.29%, p<0.01). Before treatment, the percentage of Treg cells in the NR-group (0.25±0.24%) was significantly lower than that in MMR-group and HC-group (0.57±0.29, 0.69±0.29%, p<0.01, p<0.01, respectively), although the absolute number of Treg cells did not differ. The changes in Treg% values in the PB before treatment to 6 months after treatment in the CML patients significantly increased from 0.39±0.30 to 0.60 ±0.49% (p=0.026). However, the absolute number of Treg cells significantly decreased from 37.1±18.6 to 28.3±20.0/μL (p<0.01). Similarly, comparing before and at 1 year after treatment, the absolute number of Treg cells significantly decreased from 37.1±18.6 to 17.0±14.2/μL in the CML patients (p<0.01) and from 39.2±14.7 to 14.6±6.6/μL in the MMR-group (p<0.01). The Treg% values in the MMR-group also decreased from 0.57±0.29 to 0.31±0.18% (p=0.033). However, even 6 months after treatment, the absolute number of Treg cells was significantly higher in both the CML-group (28.3±20.0/μL) and the NR-group (29.4±15.2/μL) than that in the MPN-group (14.5±10.1/μL, p<0.01, p<0.01, respectively). The absolute numbers and percentages of CD3-CD56+ NK cells and NK activity before treatment as well as after treatment did not differ between each group. However, the absolute number of CD3-CD56+ NK cells before treatment in the CML patients (427.5±424.4/μL) was significantly higher than that in the MPN patients (192.6±87.7/μL, p=0.022). The NK activity before treatment in the MMR-group (26.5±20.5%) was significantly lower than that in the HC-group (41.7±4.1%, p<0.01), whereas the NK activity increased with time and was no longer different from that in the HC-group.Conclusions: The absolute number of Treg cells in the CML patients decreased remarkably after treatment. The absolute number and the percentage of Treg cells decreased remarkably after treatment especially among the CML patients in the MMR-group. Although the number of NK cells did not change in the MMR-group compared to that in the HC-group, the NK activity was significantly lower and tended to approach the value of the normal control group after treatment. These data suggest that patients with CML have a more profound remission by treatment with a TKI resulting in decreased levels of Treg cells and increased NK activity. Immunological control via T cells and NK cells is very important in the treatment of CML. DisclosuresSuzumiya:Chugai-Roche: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Pfizer: Research Funding; SymBio: Research Funding; Ono: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Shire Japan: Speakers Bureau; Taiho: Research Funding, Speakers Bureau; Toyama Chemical: Research Funding; Celltrion: Research Funding; Takeda: Research Funding, Speakers Bureau; Zenyaku Kogyo: Consultancy; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sumitomo Dainioppon: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Ohtsuka: Speakers Bureau.

Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers.

Type 1 diabetes is an autoimmune disease, in which pancreatic β cells are destroyed by autoreactive T cells in genetically predisposed individuals. Serum beta cell autoantibody specificities have represented the mainstay for classifying diabetes as autoimmune-mediated and for stratifying risk in first-degree relatives. In recent years, approaches were attempted to solve the difficult issue of detecting rare antigen-specific autoreactive T cells and their significance to etiopathogenesis such as the use of the MHC multimer technology. This tool allowed the specific detection of increased percentages of GAD65 autoreactive T cells by means of HLA A*02:01 GAD65 AA 114–122 pentamers in newly diagnosed diabetics. Here we provide evidence that GAD65 AA 114–122 pentamers can depict a GAD65 AA114-122 peptide expandable population of functionally and phenotypically skewed, preliminary characterized CD3-CD8dullCD56+ ‘memory-like’ NK cells in PBMC of newly diagnosed diabetics. Our data suggest that the NK cell subset could bind the HLA class I GAD65 AA 114–122 pentamer through ILT2 inhibitory receptor. CD107a expression revealed increased degranulation of CD3-CD8dullCD56+ NK cells in GAD65 AA 114–122 and FLU peptide expanded peripheral blood mononuclear cells of diabetics following GAD65 AA 114–122 peptide HLA A*02:01 presentation in respect to the unpulsed condition. CD107a expression was enriched in ILT2 positive NK cells. As opposite to basal conditions where similar percentages of CD3-CD56+ILT2+ cells were detected in diabetics and controls, CD3-CD56+CD107a+ and CD3-CD56+ILT2+CD107a+ cells were significantly increased in T1D PBMC either GAD65 AA 114–122 or FLU peptides stimulated after co-culture with GAD65 AA 114–122 pulsed APCs. As control, healthy donor NK cells showed similar degranulation against both GAD65 AA 114–122 pulsed and unpulsed APCs. The pathogenetic significance of the CD3-CD8dullCD56+ ‘memory-like NK cell subset’ with increased response upon secondary challenge in diabetics remains to be elucidated.

Natural Killer Group 2A Expressed on Both Peripheral CD3-CD56+NK Cells and CD3+CD8+T Cells Plays a Pivotal Negative Regulatory Role in the Progression of Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

To explore the role of surface receptors natural killer group 2A (NKG2A) and natural killer group 2D (NKG2D) on CD3+CD8+T cells and CD3-CD56+NK cells in the progression of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF), we measured the expression of NKG2A and NKG2D on the surface of these 2 types of circulating cells by flow cytometry in 3 groups. One group consists of 36 patients with chronic hepatitis B (CHB), another one consists of 22 patients with HBV-related ACLF, and the last one has 12 normal controls (NC). The experimental result indicated that there was no significant difference in the proportion of CD3+CD8+T cells in total lymphocytes between the 3 groups. However, the percentage of CD3-CD56+NK cells in ACLF group was evidently higher than that in the CHB group (P < 0.05). In addition, the expression of NKG2D on CD3+CD8+T cells in the ACLF group was significantly lower than that in the CHB group (P < 0.05), but there were no statistically significant differences in its percentages on CD3-CD56+NK cells between the 3 groups. The expression of NKG2A on CD3+CD8+T cells in the ACLF group was significantly higher than that in the NC group (P < 0.05), and on NK cells was significantly higher than that in the CHB group (P < 0.05) and NC group (P < 0.01). The increase in ratios of NKG2A to NKG2D on CD3+CD8+T cells and CD3-CD56+NK cells in the ACLF group was significantly more than that in the CHB group and NC group. The results indicate that the imbalance between NKG2A and NKG2D may contribute to the progression of HBV-related ACLF mediated by CD3-CD56+NK cells and CD3+CD8+T cells. Compared with NKG2D, NKG2A expressed on both peripheral CD3-CD56+NK cells and CD3+CD8+T cells plays a more pivotal negative regulatory role in the progression of HBV-related ACLF.

Administration of Dexamethasone during Initial Exposure to Factor VIII Prevents the Development of Anti-Factor VIII Antibodies and Increases the Proportion of Thymic but Not Splenic Regulatory T Cells in the Exon 16 Knockout C57Bl6 Hemophilia Α Mouse Model

Background: The most severe complication of hemophilia A (HA) treatment is the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). The presence of immunological 'danger signals’ during initial exposure to FVIII may increase the risk of developing inhibitors. Promoting an anti-inflammatory environment during initial exposure to FVIII may therefore prevent inhibitor development in HA patients.Glucocorticoids are used clinically for their immunosuppressive and anti-inflammatory properties. There has also been evidence to suggest that these agents have the ability to induce regulatory T cells (Tregs): a CD4+CD25+FoxP3+T cell subset involved in the maintenance of tolerance to self-antigens.Previous work in our laboratory showed that administration of the glucocorticoid dexamethasone (Dex) during initial FVIII exposure renders tolerance to FVIII in exon 17 knockout (E17KO) HLADRB1*1501 C57Bl6/S129 HA mice. When treated with recombinant human FVIII (rhFVIII) this model has an anti-FVIII antibody incidence of 30%. In contrast, the E16KO C57Bl6 HA mouse model has 100% antibody incidence when treated with rhFVIII. Using this second model provides a method for validating our treatment protocol in an animal model with no inherent tolerance to FVIII and facilitates future mechanistic studies due to its homogeneous genetic background.Aims: To assess the ability of Dex administration during initial FVIII exposure to reduce the FVIII immune response in a HA mouse model with high propensity for inhibitor development, and enumerate lymphocyte subsets in the spleen and thymus to examine possible mechanisms of this treatment.Methods: E16KO C57Bl6 HA mice received Dex (75 μg, intraperitoneally) and rhFVIII (6 IU, intravenously) (rhFVIII+Dex Group) or rhFVIII alone (rhFVIII Group) for five consecutive days. Five weeks later, blood was collected via cardiac puncture. Plasma anti-FVIII antibody titres and FVIII inhibitory activity were determined using an anti-FVIII immunoglobulin G (IgG) ELISA and Bethesda assay respectively. Statistical comparisons were calculated using the Fisher’s exact and Mann-Whitney U tests.To elucidate early effects of the treatment, E16KO C57Bl6 mice received rhFVIII, Dex, rhFVIII+Dex, or HBSS for five consecutive days. Three days later the spleen and thymus were harvested. The percentages of splenic B cells (CD19+) and Tregs (CD4+CD25+FoxP3+) as well as thymic Tregs (CD4+CD8-CD25+FoxP3+) were quantified by flow cytometry. Statistical comparisons were calculated using a 2-tailed Student’s t-test.Results: Five weeks after treatment, 77% of mice in the rhFVIII+Dex Group vs 100% of mice in the rhFVIII Group developed detectable anti-FVIII IgG (p=0.0485). Furthermore, mice in the rhFVIII+Dex Group had overall lower anti-FVIII IgG titres (p=0.0063) and lower inhibitory activity (p=0.0783) than mice in the rhFVIII Group.Examination of the spleen three days after Dex treatment showed a decrease in the percentage of CD19+ cells in comparison to HBSS (41% vs 54%, p=0.0152). The same effect was observed with Dex+rhFVIII in comparison to rhFVIII (48% vs 54%, p=0.0489) or HBSS (48% vs 54%, p=0.0212). No significant changes were detected in the percentage of CD4+CD25+FoxP3+splenocytes across treatment groups.In the thymus, Dex caused a decrease in the percentage of CD4+CD8+ cells (42% vs 76%, p=0.0184) and an increase in the percentage of CD4-CD8+ cells (24% vs 7%, p=0.0107) in comparison to HBSS. The same effects were observed with Dex+rhFVIII in comparison to rhFVIII (40% vs 72%, p=0.0204; 20% vs 8%, p=0.0368) or HBSS (40% vs 76%, p=0.0137; 20% vs 7%, p=0.0266). The percentage of CD4+CD8- cells remained unchanged across treatment conditions. Dex caused an increase in the percentage of thymic CD4+CD8-CD25+FoxP3+ cells when compared to HBSS (11% vs 4%, p=0.0188). The same effect was observed with Dex+rhFVIII in comparison to rhFVIII (12% vs 5%, p=0.0006) or HBSS (12% vs 4%, p=0.0001).Conclusions: Administration of Dex during initial FVIII exposure reduces the anti-FVIII immune response in a HA mouse model with high propensity for inhibitor development. The effect is accompanied by a decrease in the percentage of splenic B cells and thymic CD4+CD8+ cells, and an increase in the percentage of thymic CD4-CD8+ cells and Tregs. These findings suggest potential mechanisms whereby glucocorticoid administration results in tolerance to FVIII in HA mice. DisclosuresMoorehead:Baxter: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria. Reipert:Baxter Innovation GmbH: Employment. Hough:Bayer: Research Funding. Lillicrap:Bayer: Research Funding; Baxter: Research Funding; Biogen-Idec.: Research Funding; CSL-Behring: Research Funding.

Range-reference determination of lymphocyte subsets in Moroccan blood donors

Information on lymphocyte populations (T, B, and Natural killer cells) and subpopulations (CD4 and CD8) in Morocco is scarce if not inexistent. To establish a reference value of these cells in 242 Moroccan young adult blood donors by flow cytometry. Smokers had significantly higher total leukocyte count (p < 0.001), total lymphocyte count (p < 0.0001) and higher CD3+CD4+ cells (p < 0.0001). The percentage of CD3-CD56+ subsets was affected by smoking (p < 0.01). Our analysis positively correlate with previous observations of an increase of absolute CD4+ T cells, with no changes in other lymphocyte subset cells in smokers. The lymphocyte subpopulation distributions for all antigens were found to be similar to those reported in Saudi and Italian adults, while higher levels were reported for the same gender in other countries, especially Ghana and Kuwait. The international classification standards of the HIV-infected subjects according to their rates of CD4 are applicable to the present study's population.

The influences of thymectomy on the fas expression and T Cell subsets in peripheral blood lymphocyte from patients with myasthenia gravis

Objective To investigate the influences of thymectomy on the Fas expression and T cell subsets in peripheral blood lymphocyte (PBL) from patients with myasthenia gravis ( MG), so as to make sure whether Fas-mediated apoptosis is involved in the mechanism of thymectomy in MG therapy. Methods We analyzed CD4 、CD8 and Fas expressions in PBL from 17 patients with MG and 13 healthy controls using flow cytometric analysis. Results The percentage of CD4-CD8+ cells in PB of MG patients without thymectomy was significant higher, while the percentage of CD4-CD8-8-cells was significant lower, compared with controls ( P ＜0. 05 ). The percentage of CD4-CD8 + cells in PBL of MG patients with thymectomy was significant lower than the ones without thymectomy( P ＜ 0. 05 ). The percentages of Fas + and CD8 + Fas + cells in PBL of MG patients without thymectomy, as well as the percentage of Fas + cells in PBL of MG patients with thymectomy, were significant higher than that of controls ( P ＜ 0. 05 ). The percentages of Fas + , CD4 + Fas + and CD8 + Fas + cells in PBL of MG patients with thymectomy were lower than the ones without thymectomy, but the differences were not significant ( P ＞ 0. 05 ). Conclusion T cell subsets in PBL from patients with MG is abnormal, compared with healthy control. After thymectomy, this abnormality could be revised. Fas expression on PBL of patients with MG is increased, which most likely reflects lymphocyte activation in vivo, as well as increased CD8 + T cell-mediated cytotoxic effect. Fas expression on PBL is decreased after thymectomy, which may be involved in the mechanism of thymectomy in MG therapy. Key words: Myasthenia gravis; Fas antigen; T cell subsets; Thymectomy

PTU-054 Peripheral blood and intrahepatic natural killer cells in chronic hepatitis C: relation to disease activity and hepatic fibrosis

<h3>Introduction</h3> Cellular immune responses are thought to play a key role in the pathogenesis of hepatitis C virus (HCV)-related liver damage. Recently, increasing attention has been drawn towards components of the innate immune system to HCV, including natural killer (NK) cells. The present work was designed to study peripheral blood and intrahepatic NK cells in patients with chronic hepatitis C in relation to disease activity and severity of hepatic fibrosis. <h3>Methods</h3> Fifteen patients with untreated chronic hepatitis C (CHC) and 12 healthy subjects were included in the current study. The NK and NKT cells in fresh whole blood samples were identified using two-colour flow cytometry as CD3-CD56+ and CD3+CD56 cells, respectively, and the results were expressed as percentages of the total lymphocyte count. Liver biopsies were taken from all patients and the specimens were evaluated as regards the histological activity grade and fibrosis stage according to METAVIR scoring system and for the presence and grade of steatosis. Immunohistochemical staining was done using antibodies against CD56 and smooth muscle actin (SMA) for detection of intrahepatic NK cells and activated hepatic stellate cells (HSCs), respectively. A semi-quantitative method was used to score the intensity of immunostaining. <h3>Results</h3> The percentages of CD3-CD56+ NK cells and CD3+CD56+ NKT cells in peripheral blood showed significant decreases in patients with CHC compared with healthy subjects (p&lt;0.01) and was positively correlated with the intensity of intrahepatic NK cells (p=0.001). The CD56+ NK cell infiltrate was found to be absent or minimal in about 70% of the liver biopsies of patients with CHC. Patients presented with chronic fatigue showed significantly lower percentages of circulating NK and NKT cells and intensity of intrahepatic NK cells than patients who were asymptomatic (p&lt;0.05). The percentages of peripheral blood NK cells and NKT cells and the intensity of intrahepatic NK cells showed significant inverse correlations with serum HCV RNA levels, steatosis grade, METAVIR fibrosis stage and intensity of activated HSCs (p&lt;0.05) and statistically insignificant correlations with serum levels of aminotransferases and the histological activity grade (p&gt;0.05). <h3>Conclusion</h3> The deficiency of peripheral blood and intrahepatic NK cells in patients with chronic hepatitis C may provide a mechanism for immune suppression resulting in viral persistence, disease chronicity and progression of hepatic fibrosis. Restoration of the NK cell population may be one of the potential manipulations for resolution of HCV infection and for therapeutic modulation of hepatofibrogenesis.

Cholera toxin transiently inhibits porcine T cell proliferation in vitro

Cholera toxin (Ctx) is an important mucosal adjuvant with potential experimental applications in pigs. However, little is known about the direct effects of Ctx on porcine immune cells. Therefore, we analysed the influence of Ctx on mitogen-induced lymphocyte proliferation. Ctx inhibited peripheral blood mononuclear cell (PBMC) proliferation with an IC50 of 34+/-17 ng/mL. This inhibition was not due to increased cell death. Lymphoblast formation in cultures stimulated with concanavalin A and Ctx was decreased at 24 h, but had reached the levels of control cultures again at 72 and 120 h, indicating that suppression was transient. Analysis of T cell subsets revealed that Ctx treatment specifically reduced the percentage of CD4-CD8+ and gammadelta T cells, whereas the proportion of CD4+CD8- increased. Furthermore, Ctx caused secretion of IL-10 by PBMC cultures, but depressed TNFalpha secretion.

Effect of in ovo administered reovirus vaccines on immune responses of specific-pathogen-free chickens.

We investigated the effect of in ovo administered reovirus vaccines on the immune responses of specific-pathogen-free chickens. T-cell mitogenic responses to concanavalin A were numerically lower at 9 and 12 days of age and significantly lower at 6 days of age in birds vaccinated with a commercial reovirus vaccine compared with unvaccinated birds or birds vaccinated with an experimental reovirus-antibody complex vaccine. There were no significant differences in proportions of subpopulations of helper (CD4+CD8-) or cytotoxic (CD4-CD8+) T cells except at 12 days of age, when the percentages of CD4-CD8+ cells in the two vaccinated groups were statistically higher than in the nonvaccinated group. B-cell populations were not different among vaccine groups except at 9 days of age, when the vaccinated groups had the highest level of B cells. This commercial reovirus vaccine should not be given in ovo to embryos having little or no maternal antibody, otherwise immunosuppression may occur in the chicks. The addition of the antibody complex to the vaccine prevented this T-cell immunosuppression.

Early Exposure to Genistein Exerts Long-Lasting Effects on the Endocrine and Immune Systems in Rats

Although the immunologic effects of endogenous and synthetic estrogens are well studied, few studies have examined the hormonal effects of phytoestrogens (i.e., plant-derived estrogens) on the immune system. The primary goal of this study was to compare the effects of perinatal exposure with life-long exposure to genistein, an estrogenic compound in soy, on the endocrine and immune system in adulthood. Pregnant female rats were exposed to no, low (5 mg/kg diet), or high (300 mg/kg diet) genistein diets throughout gestation and lactation. At weaning, male offspring exposed to genistein perinatally were either switched to the genistein-free diet or remained on the genistein-dosed diets. At 70 days of age, immune organ masses, lymphocyte subpopulations, cytokine concentrations, and testosterone concentrations were assessed in male offspring. Data were analyzed based on the diets that males were exposed to during gestation and lactation because life-long exposure to genistein had no additional effect on any of the dependent measures. Relative thymus masses were greater among males exposed to the high genistein diet than among males exposed to no genistein. Although the proportions of splenic and thymic CD4+ T cells were not altered by genistein, the percentages of CD4+CD8+ thymocytes, CD8+ splenocytes, and total T cells in the spleen were higher and the percentages of CD4-CD8- thymocytes were lower among males exposed to genistein than among males not exposed to genistein. Synthesis of interferon-gamma (IFN-gamma) was marginally higher and testosterone concentrations were lower among genistein-exposed than genistein-free males. These data illustrate that exposure to genistein during pregnancy and lactation exerts long-lasting effects on the endocrine and immune systems in adulthood. Whether exposure to phytoestrogens during early development affects responses to infectious or autoimmune diseases, as well as cancers, later in life requires investigation.