Surface Of Peripheral Blood Research Articles

Relationship Between the Expression of PD-1 and CTLA-4 on T Lymphocytes and the Severity and Prognosis of Sepsis.

The study aimed to investigate the relationship between the expression of PD-1 and CTLA-4 on the surface of peripheral blood T lymphocyte subsets in patients with sepsis and the severity and prognosis of the disease. The study included patients with sepsis who were admitted to the intensive care unit. The expression of PD-1 and CTLA-4 on T lymphocyte subsets was detected by flow cytometry, and the severity of sepsis was assessed using the SOFA score. The expression of PD-1 on CD4+T cells, PD-1 on Tregs, and CTLA-4 on Tregs increased with the severity of the disease (P<0.05). Multivariate logistic regression analysis showed that PD-1 expression on CD4+T cells, CTLA-4 expression on Tregs, and the SOFA score were independent risk factors for 28-day mortality in patients with sepsis (P<0.05). The area under the curve of the SOFA score combined with the expression of PD-1 on CD4+T cells and CTLA-4 on Treg cells was significantly higher than any single indicator (P<0.05). Patients with high expression of PD-1 on CD4+T cells (>31.25%) and CTLA-4 on Tregs (>12.64%) had a lower 28-day survival rate (P<0.05). The increased expression of PD-1 and CTLA-4 on CD4+T cells and Tregs is significantly associated with the severity and prognosis of sepsis patients. The combination of the SOFA score and the expression of PD-1 on CD4+T cells and CTLA-4 on Tregs can further improve the prognostic predictive value. These findings may be promising biomarkers for prognostic assessment, risk stratification, and identification of immunosuppression in patients with sepsis.

Human transitional and IgMlow mature naïve B cells preserve permissive B-cell receptors.

The level of immunoglobulin M (IgM) displayed on the surface of peripheral blood B cells exhibits a broad dynamic range and has been associated with both development and selection. To determine whether IgM surface expression associates with distinct immunoglobulin heavy‐chain (IGH) repertoire properties, we performed deep IgM sequencing of peripheral blood transitional and mature naïve B cells in the upper and lower quartiles of surface IgM expression for 12 healthy donors. Mature naïve B cells within the lowest quartile for surface IgM expression displayed more diverse IGH features including increased complementarity‐determining region 3 length, IGHJ6 segment usage and aromatic amino acids compared with mature naïve B cells with high surface IgM. There were no differences between IGH repertoires for transitional B cells with high or low surface IgM. These findings suggest that a selection checkpoint during progression of transitional to mature naïve B cells reduces the breadth of the IGH repertoire among high surface IgM B cells but that diversity is preserved in B cells expressing low levels of surface IgM.

A New Human B-Lymphocyte Surface Antigen (BL 2) Detectable by a Hybridoma Monoclonal Antibody: Distribution on Benign and Malignant Lymphoid Cells

A hybridoma-derived monoclonal antibody, produced by immunization with the Burkitt's tumor-derived B-lymphoblastoid cell line, B35M, was previously shown to detect a 68,000 dalton surface membrane protein, BL2, on the surface of peripheral blood B cells, which is absent from thymocytes, T cells, and granulocytes. In this study, we investigated the expression and distribution of BL2 on benign and malignant human lymphoid cells. Indirect immunofluorescent assay with this monoclonal antibody demonstrated that BL2 is expressed by cells within the fetal liver and by a variable proportion of lymph node, tonsil, and spleen B cells, but not by T cells. The neoplastic cells isolated from 18 T-cell malignancies were BL2- . BL2 was was heterogeneously expressed by a variable proportion of the malignant cells in 29/32 cases of B-chronic lymphocytic leukemia and 33/38 cases of B-cell lymphomas, but appeared to be lost in the terminal stages of B-cell differentiation, as myeloma plasma cells were BL2- . BL2 expression was not limited to B cells of a particular surface immunoglobulin isotype. Immunofluorescent staining for BL2 in cryostat tissue sections demonstrated that the majority, but not all, germinal center and interfollicular Ia+ (non-T) cells are BL2+. These findings suggest that BL2 is a B-cell lineage-specific differentiation marker that may be useful in the study of B-cell ontogeny and in defining subgroups of the B-cell malignancies.

Expression of HLA-DP in patients with neuromyelitis optica spectrum disorders

Objective: To investigate the effect of HLA-DP gene expression on the susceptibility and disease status of neuromyelitis optica spectrum disorders (NMOSD). Methods: A total of 86 NMOSD patients (52 in acute phase and 34 in remission phase), 52 multiple sclerosis (MS) patients (20 in acute phase and 32 in remission phase) diagnosed in Department of Neurology, the Third Affiliated Hospital of Sun Yat-sen University and 29 healthy controls were enrolled prospectively. Genotyping of HLA-DP was performed. The expression levels of HLA-DP molecules in peripheral blood B cells and monocytes were measured by flow cytometry. The transcription levels of HLA-DPB1 mRNA in peripheral blood mononuclear cells (PBMC) were measured by real time-PCR. The results were compared among different groups Results: There was no statistically significant difference of the distributions of HLA-DPB1*0501/HLA-DPB1*0501, HLA-DPB1*0501/X and X/X genotypes and the frequencies of allele of HLA-DPB1*0501 among NMOSD, MS patients and healthy controls (P=0.96 and 0.71, respectively). The expression levels of HLA-DP on the surface of B cells in NMOSD patients, especially in remission phase patients, were significantly higher than those in healthy controls(212±328 and 374±394 vs 55±57, P=0.049 and 0.002, respectively). The expression levels of HLA-DP on the surface of monocytes in NMOSD patients in acute phase were significantly higher than those in healthy controls(158±175 vs 65±90, P=0.025). The transcription levels of PMBC HLA-DPB1 mRNA in acute phase and remission phase of NMOSD patients were significantly higher than those in healthy controls (3.0±1.4 and 2.9±1.3 vs 1.5±1.4, P=0.000 and 0.003, respectively). The expression levels of HLA-DP molecules on the surface of peripheral blood B cells and monocytes and the transcription levels of PMBC HLA-DPB1 mRNA in MS patients at the acute and remission stages were not significantly different from those in healthy controls. The expression levels of HLA-DP molecules on the surface of B cells in patients with HLA-DPB1*0501/HLA-DPB1*0501, HLA-DPB1*0501/X and X/X genotypes were statistically different (P=0.017). Conclusion: HLA-DP gene transcription and molecular expression levels in antigen presenting cells may affect the susceptibility and disease status of NMOSD patients, while HLA-DPB1*0501 allele may affect the transcription and molecular expression levels of HLA-DP gene in antigen presenting cells.

Relationship between viral load and expression of CD226 on the surface of T follicular helper cells in patients with chronic hepatitis C

Objective To explore the relationship between viral load and expression of CD226 on the surface of peripheral blood T follicular helper cells (Tfh) in patients with chronic hepatitis C (CHC). Methods One hundred and thirty-five CHC patients hospitalized at Wuxi Fifth People′s Hospital from March 2015 to April 2017 were collected, and another 30 healthy blood donors were set as healthy control group. CHC patients were divided into two groups based on hepatitis C virus (HCV) RNA level, with 49 cases (36.3%) in low viral load group and 86 cases (63.7%) in high viral load group. Expression of CD226 on the surface of peripheral blood Tfh cells, Tfh cells, interleukin (IL)-21 and HCV specific cytotoxic lymphocyte (CTL) levels of two patient groups were compared. Categorical data were compared with chi-square test and normally distribute continuous data were compared with t test. Correlations between different factors were analyzed by Pearson correlation analysis. Results Expression of CD226 on the surface of peripheral blood Tfh cells in 135 cases of CHC patients was (77.69±5.42)%, which was lower than that of healthy control ([90.06±5.83]%), and the difference between the two groups was significant (t=7.541, P<0.01). The CD226 expression on the peripheral blood Tfh cells in low viral load group was (88.75±6.68)%, which was higher than that of high viral load group ([69.23±5.86]%), and the difference between the two groups was significant (t=19.232, P<0.01). The viral load was negatively correlated with Tfh cell surface CD226 expression (r=-0.705, P<0.01). The peripheral blood Tfh cell level in 135 CHC patients was higher than that of healthy control, and the difference between the two groups was significant (t=13.878, P<0.01). The peripheral blood Tfh cell level in low viral load group was higher than that in high viral load group, and the difference between the two groups was significant (t=26.993, P<0.01). The IL-21 level of 135 CHC patients was lower than that of healthy control ([70.35±1.6]%), and the difference between the two groups was significant (t=18.322, P<0.01). The IL-21 level in peripheral blood of low viral load group was higher than that of high viral load group, and the difference between the two groups was significant (t=84.54, P<0.01). HCV specific CTL level in peripheral blood of low viral load group was higher than that of high viral load group, and the difference between the two groups was significant (t=29.869, P<0.01). The viral load was negatively correlated with levels of HCV specific CTL (r=-0.734, P<0.01). Conclusions In patients with chronic hepatitis C, different levels of viral load can result in different levels of CD226 expression on the peripheral blood Tfh cells. Patients with low viral load has high CD226 expression on Tfh cell surface, resulting in rise of Tfh cell level, IL-21 level and HCV specific CTL level. Key words: Hepatitis C, chronic; T-lymphocytes, cytotoxic; T-lymphocytes, follicle helper; CD226; Interleukin-21

Role of Moesin in Murine Hematopoiesis

Abstract 1338 Background:Moesin, a membrane-cytoskeleton linker protein, functions in microtubule formation, in membrane ruffling, and at sites of cell-adhesion. However, its role in hematopoiesis is unclear. Although a previous study demonstrated normal hematopoietic function in moesin knock-out (KO) mice (JBC 1999), the changes in blood cell counts observed in the mice have not been thoroughly analyzed. Moesin-specific antibodies are detected in the serum of approximately 30% of patients with acquired aplastic anemia (AA). Moesin is expressed on the surface of peripheral blood T cells and monocytes, but not on hematopoietic stem cells (HSCs). Our recent study demonstrated the involvement of anti-moesin antibodies in the development of AA by stimulating T cells and monocytes to secrete tumor necrosis factor-α and interferon-γ. Given that hematopoiesis is regulated by T cells and monocytes, the absence of moesin should impact hematopoiesis. The purpose of this study was to examine blood cell counts in moesin KO mice and WT mice transplanted with anti-moesin antibody producing B cells to clarify the role of moesin in hematopoiesis. Methods:Moesin KO mice (F1 heterozygous females (-/X)), which were a hybrid strain between 129/Sv and C57BL/6 (B6), were kindly provided by Professor Tsukita of Osaka University. F1 heterozygous females and B6 F1 wild-type (WT) males were interbred and the littermates were genotyped. Moesin KO mice, F2 hemizygous males (-/Y), and F3 homozygous females (−/−) were analyzed in a previous study (JBC 1999). F1 heterozygous females were backcrossed to B6 males for at least 8 generations to generate moesin KO mice in a B6 background. Moesin KO mice (12 months old) were used for hematopoietic phenotype analysis and to raise anti-moesin antibodies by immunization with moesin protein. Moesin KO male mice (-/Y) were immunized with 2×107 spleen cells containing moesin from WT B6 male mice to produce anti-moesin antibody secreting cells. Anti-moesin antibody titers were determined by Western blot analysis. To analyze the effects of anti-moesin antibodies in hematopoiesis, spleen cells (1.5×107) and bone marrow (BM) cells (1×107) containing anti-moesin antibody secreting cells from moesin KO mice were transplanted to the BM of 8-week-old WT B6 male mice conditioned with irradiation (3 Gy). Results:Moesin KO mice were born at expected Mendelian ratios and developed and grew normally. However, the body weight of moesin KO mice (n=7) was lower than that of WT mice (n=9) (26.63±2.47 g vs. 30.71±4.85 g; P=0.049). Complete blood counts of moesin KO mice (n=4) revealed leukocytopenia (WBC 12000±3096/μL vs. 19750±2582/μL; P=0.03) and macrocytic anemia (Hb 13.8±0.50 g/dL vs. 15.8±0.50 g/dL; P=0.06; MCV 51.7±1.7 fL vs. 47.6±1.3 fL, P=0.002) as compared with WT mice (n=4); platelet counts were comparable. Moesin KO mouse spleens (n=8) were heavier than those of WT mice (0.316±0.355 g vs. 0.091±0.034 g; P=0.0002) and follicle formation in spleens of moesin KO mice were obscure with increased CD3+ lymphocytes and reduced CD19+ lymphocytes as compared with WT mice. BM of moesin KO mice was normocellular and characterized by increased CD3+ lymphocytes and reduced CD19+ lymphocytes as seen in spleens of moesin KO mice. The number of lineage negative, Sca-1+, and c-Kit+ (LSK) mouse HSCs in moesin KO BM (n=4) tended to be lower than that of WT mice (n=3) (0.97±0.29% vs. 1.85±0.20%; P=0.06). Moesin KO spleen/BM cells capable of producing anti-moesin antibodies were infused into BM of WT B6 male mice (n=3), and moesin KO hematopoietic cells were engrafted on day 21. Transplanted mice showed leukocytopenia compared to WT B6 male mice transplanted with WT spleen/BM cells (n=3) (WBC 2667±333/μL vs. 7333±333/μL; P=0.0006), but anemia and thrombocytopenia were not observed. Conclusions:Moesin plays an important role in murine hematopoietic and immune system development. Anti-moesin antibody-induced leukocytopenia may result from excessive secretion of myelosuppressive cytokines from immune cells as observed in AA patients. The mechanistic details of HSC impairment in moesin KO mice are currently under investigation. Disclosures:No relevant conflicts of interest to declare.

Relationship between CTLA-4 and CD28 Molecule Expression on T Lymphocytes and Stimulating and Blocking Autoantibodies to the TSH-Receptor in Children with Graves’ Disease

The present study was performed to elucidate the relationship between CTLA-4/CD28 molecules and stimulating (TSAb) and blocking (TBAb) antibodies to the TSH-receptor (TSH-R) in Graves’ disease. CD28 and CD152 (CTLA-4) are glycoprotein molecules which provide a potent costimulatory signal for T-cell activation and proliferation via interactions with their ligands, B7.1/B7.2 molecules, which are present on the surface of antigen-presenting cells. The aim of the study was to estimate the expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152), CD28, B7.1 (CD80), and B7.2 (CD86) molecules on peripheral blood cells in patients with Graves’ disease (GD) (n = 55, mean age 15.5 ± 5.1 years) and nontoxic nodular goiter (NTNG) (n = 55, mean age 15.2 ± 4.5 years), in comparison with sex and age-matched healthy control subjects (n = 55, mean age 15.2 ± 3.9 years). The expression of the costimulatory molecules on mononuclear cells was analyzed by three-color flow cytometry using a Coulter EPICS XL cytometer. Detection of TSAb and TBAb to the TSH-R using JPO9 CHO cells in unfractionated serum was measured by a highly sensitive commercial radioimmunoassay. When compared with healthy control subjects and euthyroid patients with GD, untreated patients with GD showed a significant increase of CD152+ (p < 0.001, p < 0.001) and CD28+ (p < 0.01, NS) T lymphocytes, respectively. After 6–12 months of methimazole therapy, the percentage of these cells in the peripheral blood of hyperthyroid patients returned to normal values. In addition, patients with GD showed an increase in the percentage of both B7.1 (3.8%) and B7.2 (18.4%) molecules on activated monocytes, compared to patients with NTNG (0.5% p < 0.05, 2.5% p < 0.01, respectively) and healthy control subjects (0.2% p < 0.05, 0.8% p < 0.003, respectively). In patients with untreated GD there was a statistically significant positive correlation between the expression of CTLA-4 on the surface of peripheral blood T cells and the index of TSAb antibodies (R = 0.54, p < 0.001) as well as a negative correlation with TBAb antibody titer (R = –0.58, p < 0.001). However, no such correlations were noted with regard to CD28 and anti-TPO, anti-TG, and TRAb antibodies. We conclude that changes in the expression of costimulatory molecules on the surface of peripheral blood T cells and their significant relationship with the level of antithyroid antibodies indicate an involvement of these molecules in the pathogenesis of GD.

Expression of intercellular adhesion molecule-1 on the surface of peripheral blood and decidual lymphocytes of women with pregnancy-induced hypertension

Objective: If overexpression of intercellular adhesion molecule-1 (CD54) on lymphocytes exists it could have important implications for the pathophysiology of pregnancy-induced hypertension (PIH). Study design: CD54 “in vitro” expression (described as (%) of CD54+cells and CD54 mean fluorescence intensity, MFI) on the peripheral blood and decidual lymphocytes of pregnant with pre-eclampsia (PE) ( n=16), transient hypertension (TH) ( n=12), and controls ( n=9). Results: The percent (%) of CD54+peripheral blood lymphocytes, CD54 MFI on them and CD54 MFI on decidual lymphocytes were increased especially in PE. During PE the (%) of CD54 decidual lymphocytes correlated negatively with platelet count. In TH the positive correlation between the blood pressure and the (%) of CD54 peripheral blood lymphocytes as well as CD54 MFI on decidual lymphocytes were found. Conclusions: (1) PIH, especially PE, is accompanied by overexpression of intercellular adhesion molecule-1 (ICAM-1) on peripheral blood and decidual lymphocytes studied “in vitro”, (2) Some of the parameters studied seem to correlate with clinical markers of PIH intensity but this fact needs further investigations using larger subject groups.

Molecular cloning of the gene coding for the mouse T-cell antigen CD7.

The CD7 antigen is an M{sub r}40000 glycoprotein expressed on the surface of peripheral blood T lymphocytes and thymocytes. As one of the earliest surface antigens to appear on the cells of T-cell lineage, CD7 has been used as a valuable marker for healthy and malignant T cells. In an attempt to elucidate functionally important structures of CD7 by comparison between different species, we isolated mouse cDNAs encoding the mouse homologue of human CD7. Mouse Cd7 showed a 66% identity with human CD7 and six highly conserved regions were identified, although the four repeats of the stalk region sequence could not be identified. In the present study, we report the genomic organization of the mouse Cd7 gene. 9 refs., 3 figs., 1 tab.

T-Cell and macrophage activation in experimental autoimmune neuritis and Guillain-Barr� syndrome

Evidence implicating cellular immune responses in the pathogenesis of experimental autoimmune neuritis (EAN) and Guillain-Barré syndrome (GBS) is reviewed. In EAN the decisive role of T-lymphocytes in the initiation of immune-mediated nerve damage has been firmly established by adoptive transfer experiments. Macrophages but not Schwann cells express major histocompatibility complex class II gene products in situ and hence may function as antigen presenters. Macrophages are crucial in the amplification and effector phase and damage the myelin sheath by phagocytic attack and release of inflammatory mediators such as toxic oxygen radicals, arachidonic acid metabolites, complement, or hydrolases. Macrophage activation in EAN is achieved by interferon-gamma. Attempts to detect specific sensitization of T-lymphocytes to nerve antigens in patients with GBS have so far been unsuccessful. However, circulating activated T cells can be found in patients with GBS, as evidenced by augmented expression of HLA-DR antigen, the transferrin receptor, and the interleukin-2 receptor on the surface of peripheral blood T cells, and by increased serum concentrations of interleukin-2 and the soluble interleukin-2 receptor. In addition, we present data indicating macrophage activation in GBS.