Expression Of Lymphocyte Surface Markers Research Articles

Impaired surface marker expression in stimulated Epstein-Barr virus transformed lymphoblasts from Barth Syndrome patients

Primary B lymphocytes rapidly respond to lipopolysaccharide (LPS) and cytosine linked to a guanine by a phosphate bond deoxyribonucleic acid (CpG DNA) stimulation to promote adaptive immune function through increased surface marker expression. Here we examined expression of surface markers in LPS and CpG DNA stimulated Epstein-Barr virus transformed B lymphoblasts from control and BTHS patients with different mutations. The percentage of cluster of differentiation (CD) positive cells including CD38 + , CD138 + , CD80 + surface expression and programmed cell death protein 1 (PD1 +) surface expression was similar between control and BTHS lymphoblasts incubated plus or minus LPS. The percentage of CD24 + , CD38 + and CD138 + cells was similar between control and BTHS lymphoblasts incubated plus or minus CpG DNA. CD27 + surface marker expression was reduced in both BTHS lymphoblasts and controls incubated with CpG DNA and PD1 + surface marker expression was higher in BTHS cells compared to controls but was unaltered by CpG DNA treatment. Thus, Epstein-Barr virus transformed control and BTHS lymphoblasts fail to increase selected surface markers upon stimulation with LPS and exhibit variable surface marker expression upon stimulation with CpG DNA. Since B lymphocyte surface marker expression upon activation is involved in B cell proliferation and differentiation, cell–cell interaction and the adaptive immune response, we suggest that caution should be exercised when interpreting immunological data obtained from Epstein-Barr virus transformed BTHS cells. Based upon our observations in control cells, our conclusions may be more broadly applicable to other diseases which utilize transformed B lymphocytes for the study of immune biology.

Effects of frequent sweetener intake on murine splenic T lymphocyte surface marker expression

Metabolic and immunological alterations are common in chronic conditions, such as diabetes, which promotes low‐level chronic inflammation. The use of non‐nutritive sweeteners has increased not only in patients suffering from this illness, but in the general population. The effects of these compounds on the immune system are not well known. The objective of this study was to determine the effects of the frequent consumption of sucrose and stevia on T lymphocyte surface marker expression. 14 week‐old BALB/c mice were orally administered either sucrose or stevia in drinking water for 6 weeks. Subsequently, splenic T cells were purified and analyzed by flow cytometry using the following surface markers: CD3, CD4, CD8, CD25 and CD69. Our results showed an increase in CD4+ T cells of the sucrose group, compared to control and stevia groups. We observed an increase in CD8+ T cells in the sucrose and stevia groups. In contrast, we found no change in CD4+/CD25+ T cells among groups, but we observed a decrease in the percentage of CD4+/CD69+ T cells in the sucrose and stevia groups. Our data suggests that the frequent use of sweeteners modifies the frequency and phenotype of T cell subsets in the spleen.

Lymphocyte’s Activation and Apoptosis After Coronary Artery Bypass Graft: A Comparative Study of Two Membrane Oxygenators–One with and Another without a Venous-Arterial Shunt

Newer oxygenators with the latest technologies are designed to attenuate the immune response, including lymphopenia, prompted by cardiopulmonary bypass (CPB) in cardiac surgery. We evaluated the effect of CPB, comparing an oxygenator with a venous-arterial shunt and a conventional oxygenator with regard to lymphocyte's early activation and apoptosis induction and its implications in post-CPB lymphopenia. Patients undergoing coronary artery bypass graft surgery with CPB, using either a conventional oxygenator or one with a venous-arterial shunt, had blood samples drawn at anesthetic induction (baseline); the beginning and end of the CPB; and at 6, 12, and 24 hours after surgery. Analysis by flow cytometry was undertaken to assess the expression of lymphocyte surface markers (CD3+, CD25+, CD26+, CD69+) and apoptosis (annexin V). Twenty patients were studied; 10 used a conventional oxygenator, and 10 used an oxygenator with venous-arterial shunt. Postoperative lymphopenia (50% decrease), 35% increased expression of CD69+, and 56% decrease in annexin V were significant comparing baseline to 24 hour value, similarly in both groups. Early activation (expression of CD69+) and degree of apoptosis (expression of annexin V) of lymphocytes after CBP in cardiac surgery was similarly observed in both types of oxygenators. The observed lymphopenia after CPB does not appear to be secondary to apoptosis.

Deficiency of the expression of CD45RA isoform of CD45 common leukocyte antigen in CD4+ T lymphocytes in children with infantile cholestasis

The immunological background of the pathological changes that appear in infantile cholestasis (infections, inflammatory process in the liver) is largely unknown. With the use of double color flow cytometry, we assessed the distribution of functionally different lymphocyte subpopulations in the peripheral blood of 29 infants with extra and intra-hepatic cholestasis (12 and 17 patients, respectively), aged from 1 to 8.6 months. Control group consisted of 15 age-matched, healthy infants. We examined: (1) the expression of CD3, CD4, CD8, CD19 lymphocyte surface receptors; and (2) the distribution of lymphocyte subsets with distinctive surface Ag characteristics of ‘naive’ (CD45RA+) and ‘memory’ (CD45RO+) cells in both CD4+ and CD8+ cell populations. The surface markers expression was evaluated in terms of percentage of positive cells and receptor density. The following changes in the expression of lymphocyte surface markers are described: (1) a decrease in the percentage of total CD3+, CD4+ cells but normal percentage of CD8+ cells and elevated proportion of CD19+ B cells; (2) a reduction of the proportion of ‘naive’ CD4+ lymphocytes but normal percentage of ‘naive’ CD8+ as well as ‘memory’ CD4+ and CD8+ cell subsets; (3) a decrease in density of CD3, CD4+, CD8 receptors, and D45RA isoform in a subset of ‘naive’ CD4+ cells. We conclude that deficiency of ‘naive’ CD4+ T cell subset which possess important effector and immunoregulatory functions, and low expression of certain lymphocyte receptors known to be engaged in T cell activation, possibly reflect a defect of cell mediated immunity that may account for viral and bacterial infections, often observed in infants with cholestasis.

Peyer's patches are precocious to the appendix in human development.

PP are first visible at ∼15.5 wk gestation after which there is a rapid spurt in the development and maturation of lymphoid follicles so that at any given point of time new foci of PP development are continuously formed at a rapid rate. Addition of rows of follicles results in the formation of a PP. Immature PP of younger fetuses have a spongy structure in contrast with the compact lymphoid follicles of mature PP of older fetuses. Immunocytochemical studies reveal that there is a subtle gradation in the expression of lymphocyte surface markers with increasing fetal age. Expression of antigenic markers occurs in an ordered sequence viz. HLA – DR, CD19 (B cell population), CD9 (pre-B cells), CD3 T lymphocytes, CD4 helper / inducer lymphocytes, the CD8 suppressor / cytotoxic cells and lastly, the CD57 Natural Killer cells. The antigens are expressed first on lymphocytes of PP and thereafter in those of the appendix. Our findings clearly demonstrate that the ∼5 wk fetal period from 17.5 wk to 22 wk represents a major growth phase in the development of surface markers of lymphocytes in the mucosal immune system of the gut.

Possible autosomal-dominant inheritance of prepubertal periodontitis in an extended kindred.

This study presents the clinical findings and the distribution of prepubertal periodontitis in an extended family with high prevalence of this entity. The expression of surface markers and adhesion molecules on peripheral lymphocytes were also studied. Approximately 50% of the children in this family suffered from prepubertal periodontitis. All the affected children were otherwise healthy. 2 identical twins were similarly, but not identically, affected. Detailed laboratory tests and analysis of lymphocyte surface marker expression, including CD18, were all within the normal levels. Both localized and generalized forms of prepubertal periodontitis were found. The high prevalence of prepubertal periodontitis in the 2 branches of this family, and the fact that identical twins were similarly affected, suggest a strong genetic predisposition for prepubertal periodontitis. The family pedigree is consistent with an autosomal-dominant mode of transmission. The coexistence of localized and generalized forms of the disease in sibs suggests the same genetic etiology for both entities with variability in disease expression. This variability in disease expression is further supported by the fact that 2 identical twins were not identically affected.

Collagenase and Dispase enzymes disrupt lymphocyte surface molecules

Collagenase and Dispase enzymes are often used to disaggregate tissue. In this study, we examined by flow cytometry the effects of these enzyme preparations on peripheral blood lymphocyte surface marker expression. Peripheral blood mononuclear cells (PBMC) were obtained from seven healthy volunteers. Cells were incubated with collagenase (128 U/ml, type 1 A) for 3 h and overnight and with Dispase (1.6 mg/ml, grade II) for 15 min, 30 min, 1 h, 3 h and overnight. The intensity of expression of CD3, CD4, CD8, αβ and γδ T cell receptors was decreased by 25–40% in all cases, while CD4+ and CD8+ lymphocyte populations were undetectable after treatment with Dispase. Moreover, reappearance of these surface molecules did not occur following the incubation of cells in culture medium for 3 h. The results of this study emphasise the importance of considering the influence of isolation procedures on cell characteristics.

Mononuclear cells in salivary glands of normal and isoproterenol-treated rats

The purpose of this study was to analyse the phenotypical distribution of resident cells of the mononuclear phagocyte system in rat salivary glands, and to determine whether isoproterenol induces alterations in macrophage and lymphocyte surface-marker expression. Frozen sections of gland tissues were prepared from five normal rats, and from six rats treated with 20 mg/kg isoproterenol/day for 10 days. A panel of six monoclonal antibodies was used to identify membrane markers associated primarily with monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), MHC class II (Ia) antigens (OX6), CD5-positive T lymphocytes (OX19), and rat B lymphocytes (OX33). Double-labelling techniques were used to detect the coexpression of ED1/ED2 and OX6/ED2 mononuclear cell markers in the major salivary glands. ED2- positive macrophages were predominant in all three major glands, ranging from 96 cells/0.87 mm 2 field in the parotid gland to 165 cells/0.87 mm 2 in the submandibular. OX19-positive T lymphocytes were rarely observed in submandibular and parotid glands but represented a distinguishing feature of the sublingual. Moderate numbers of ED3-positive macrophages also were detected in sublingual tissues. In the submandibular and parotid glands, isoproterenol resulted in a decrease in ED2-positive cells, but ED2-positive macrophages increased in sublingual glands with isoproterenol. Isoproterenol resulted in a decrease in MHC class II antigen expression on submandibular and sublingual mononuclear cells but an induction of Ia antigen in the parotid gland. Double labelling revealed that isoproterenol induced coexpression of ED1/ED2 markers on mononuclear cells in the submandibular glands, but ED1/ED2-positive cells were absent from other glands. However, coexpression of MHC class II markers on ED2-positive cells in the sublingual and parotid glands of normal rats was frequently observed, with isoproterenol decreasing coexpression in the sublingual gland and increasing it in the parotid. B lymphocytes were not detected in any of the glands examined. These findings indicate that important differences exist in normal resident mononuclear cell subsets among the major salivary glands of the rat. The differential effects of isoproterenol on inflammatory cells may reflect important differences in local salivary gland immunoregulation. Although salivary gland inflammation induced by isoproterenol does not appear to result from immune mechanisms, the rich population of T lymphocytes and ED3-positive macrophages, and presence of MHC class II antigens, suggest that the sublingual gland may function as an immune organ and have a role in mucosal immunity.

Lymphocyte surface marker expression on hybridomas secreting human monoclonal antibodies

The expression of human leucocyte markers on the surface of hybridoma cell lines producing human monoclonal antibodies was studied using immunofluorescence analysis (FACS). We tested 36 different hybridoma cell lines from fusions of lymphocytes of different organs of fetal and adult organisms with the mouse myeloma line P3 X63 Ag8.653 or the mouse-human heteromyeloma line CB-F7 (IgM-, IgG-, and nonproducer) with a panel of 21 murine monoclonal antibodies against human differentiation and activation antigens. CD2, 3, 4, 5, 8, 10, 23, 25 antigen and major histocompatibility complex (MHC) class II determinants could not be detected on all hybridomas analyzed. The antigens CD22, 69, 71, and 72 were expressed on few of the hybridomas tested. The majority of the cell lines carried the surface markers CD19, 20, 40, 45 as well as the plasma cell markers CD38 and O/C11. The activation antigen 4F2 was expressed on all the cell lines tested. However, a direct connection between the expression of a lymphocyte marker and the capacity for Ig production (high and low producer; Ig isotype), the origin of the lymphocytes, and the fusion cell line used could not be detected.

Flow cytometric analysis of lymphocyte subpopulations in the spleen and thymus of mice exposed to an acute immunosuppressive dose of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)

The objective of the present studies was to determine if acute exposure to an immunotoxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the expression of lymphocyte surface markers as measured by multiparameter flow cytometry. The immunotoxicity of a single oral dose of TCDD was assessed by the anti-SRBC PFC response; an ED50 of 0.74 micrograms/kg was determined. Subpopulations in the spleen and thymus of C57B1/6 mice were analyzed 2 days following exposure to 2 micrograms/kg TCDD. In addition, splenic lymphocyte subsets were examined on Days 1-4 following SRBC challenge of mice treated with 0, 2, or 5 micrograms/kg TCDD. T and B cells were identified by single parameter analysis of Thy 1.2 and Ig expression. T cell subsets were defined by dual parameter analysis of CD4 and CD8 expression. In TCDD-treated mice, the percentage and the total number of double-positive CD4+ CD8+ thymocytes were significantly decreased while the percentage but not the total number of double-negative CD4- CD8- thymocytes was significantly increased. No changes in the percentage or total number of single positive (CD4+ CD8- or CD4- CD8+) thymocyte subsets were observed. In contrast to the thymus, lymphocyte subsets in the spleen were not significantly altered in percentage or total number 2 days following acute TCDD exposure. When splenic lymphocytes were analyzed daily following SRBC challenge, Ig+, Thy 1.2+, and CD4+ CD8- subpopulations remained relatively unchanged in both control and TCDD-treated animals. A small but significant decrease in the percentage of CD4- CD8+ T cells was observed on Day 3 in mice treated with 2 or 5 micrograms/kg TCDD when compared to that of vehicle-treated mice. The total number of CD4- CD8+ splenocytes was also significantly lower in the 5-micrograms/kg group on Day 3. However, this effect appeared to result from an elevation of the CD4- CD8+ subset in the controls rather than from a reduction in the TCDD-treated groups. Double-positive (CD4+ CD8+) lymphocytes were not detected in either control or TCDD-treated spleens. These results indicate that an acute dose of TCDD which reduced the splenic anti-SRBC response by 65-80% did not cause detectable changes in major splenic lymphocyte subpopulations. This is an important finding from the standpoint of utilizing lymphocyte subset analysis to screen for potential immunotoxic effects of TCDD. Specifically, the absence of subset changes does not preclude the presence of functional immunosuppression.

Lymphocyte surface marker expression in rheumatic diseases: evidence for prior activation of lymphocytes in vivo.

Expression of major histocompatibility complex (MHC) class II and other lymphocyte activation markers on peripheral blood and synovial fluid T lymphocytes from patients with rheumatoid arthritis (RA), psoriatic arthritis, and Reiter's syndrome were measured and the mean fluorescence intensities of these antigens were assessed. Increased expression of MHC class II antigens of synovial fluid T lymphocytes is not unique to RA, though it is quantitatively greater on RA synovial fluid T cells. There was less expression of other lymphocyte activation markers (4F2, transferin receptor) and a marked discordance between the expression of these markers and the interleukin 2 receptor (IL2r). Synovial fluid T lymphocytes contain a subpopulation of larger cells expressing MHC class II and other lymphocyte activation antigens with the exception of the IL2r. Mean fluorescence intensity of CD3 and CD4 antigens on synovial fluid T lymphocytes was decreased in all three patient groups, suggesting prior in vivo exposure of synovial fluid T lymphocytes to an unknown antigen.

Modulation of lymphocyte-surface marker expression in chronic hepatitis-B by treatment with gamma-interferon

Modulation of lymphocyte-surface marker expression in chronic hepatitis-B by treatment with gamma-interferon

Lymphocyte Subpopulations and Lymphokine Production in Children with Constitutional Aplastic Anemia

The expression of lymphocyte surface markers as well as the production of interleukin-2 (IL-2) and interferon-gamma (IFN) by mitogen-stimulated peripheral blood mononuclear cells (MNC) have been studied in five children with constitutional aplastic anemia. A significantly reduced T4/T8 ratio was found and two of five patients also had a reduced percentage of B cells. One patient had a high percentage of HLA-DR positive T8+ cells, very suggestive of a high degree of circulating activated T suppressor/cytotoxic cells. IL-2 production was reduced in two patients, whereas IFN production was only reduced in one of these. The abnormalities found correlate with the duration of the bone marrow failure. The patients with the longest duration of bone marrow failure also exhibited the lowest T4/T8 ratio. No spontaneous IFN production was detected in any of the patients. There was no clinical benefit or reversal of the immune abnormalities during and following treatment with cimetidine and cyclosporine A in two patients.

In vitro modulation of purine enzyme metabolism and lymphocyte surface marker expression by thymosin fraction 5 in homosexual males

Thymosin fraction 5 (Thymosin) has numerous immunoregulatory activities including modulation of enzymes involved in lymphocyte maturation. The effect of Thymosin on the purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′ nucleotidase (5′ NT) in null and T-enriched peripheral blood lymphocytes from sexually active asymptomatic homosexual males (AS), patients with the AIDS-related symptom complex (ARC), and those with acquired immune deficiency syndrome (AIDS) was examined and compared to its effect on lymphocytes from healthy heterosexual controls. Mean ADA activity was significantly higher in null cells from fourteen AIDS patients than in five asymptomatic homosexuals, ten ARC patients, or 27 controls. Mean PNP activity was significantly elevated in null-enriched lymphocytes from ten ARC and fourteen AIDS patients compared to controls. No differences in these enzymes were found in T-enriched cells from any group. 5′NT was markedly decreased in both null and T lymphocytes in all homosexual groups relative to controls. Homosexuals had significantly elevated percentages of OKT10 positive and Ia positive lymphocytes compared to controls. Thymosin at an optimal concentration of 150 μg/ml caused significant decreases in mean ADA and PNP activity in null lymphocytes from ARC + AIDS patients along with a significant decrease in the percentage of OKT10 positive lymphocytes. No phenotypic changes were seen in AS or control lymphocytes. The data suggest that Thymosin has a maturational effect in vitro on immature T cells from symptomatic homosexuals.