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PubMed1989

[Laboratory tests for cellular immunology: flow cytometric analysis of lymphocyte subsets].

Flow cytometric analysis for lymphocyte subsets provides two kinds of data: proportion of fluorescence-positive cells (lymphocyte subsets) and the fluorescence intensity. For proportional analysis of lymphocyte subsets, lymphocytes are fractionated with a conventional gate window in scatter analysis, but if activated or enlarged lymphocytes are examined, lymphocytes should be fractionated with an extended gate window. On the other hand, fluorescence intensity of a lymphocyte subset, which represents density of cell surface antigen detected by fluorescence-conjugated specific antibody, is not measured in routine tests yet. However, data on fluorescence intensity are often more valuable than the proportions of lymphocyte subsets: e.g., decrease in fluorescence intensity, but not in proportion, of CD8+ cells in active Graves' disease, and marked decrease in fluorescence intensity of OKT4+ cells in a carrier of familial OKT 4 epitope deficiency. Furthermore, two-color flow cytometry has enabled to measure more valuable, classified subsets of lymphocytes than single color flow cytometry. For example, measurement of peripheral CD5 (Leu-1)+ B cells is very useful for diagnosis of Graves' disease and its differentiation from destruction-induced thyrotoxicosis. In conclusion, we showed that measurement of fluorescence intensity of lymphocyte subsets and two-color flow cytometry are useful for laboratory tests of lymphocyte subsets.

Flow Cytometry↗
PubMed1987

[Effect of splenectomy in gastric carcinoma surgery on cell-mediated immunity; evaluation with alteration of peripheral blood lymphocyte subsets].

Peripheral blood lymphocyte subsets were studied in 70 patients who underwent gastrectomy for gastric carcinoma. Lymphocyte subsets were analysed before and after gastrectomy by use of monoclonal antibody for cell membrane markers. There were significant differences in the gastrectomized patients with splenectomy compared to the patients without splenectomy in postoperative changes of lymphocyte subsets as follows. Decrease of OKT4 and increase of OKT8. Temporary decrease of B-1. Continuous increase of Leu-7. These results suggest that gastrectomized patients with splenectomy show a decrease in helper activity, an increase of suppressor activity of T lymphocytes and a compensatory increase of population of natural killer cells in association with suppression of natural killer activity.

Female↗
PubMed1993

CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets.

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0+ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA+ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA+ and CD45R0+ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0+ than in CD45RA+ cells. Basal DAG levels in CD45R0+ cells were found to be, on average, 60% higher than in CD45RA+ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0+ cells than in CD45RA+ cells (p = 0.015 and 0.023). The CD45R0+ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA+ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0+ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0+ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0+ T cells to mitogenic stimuli compared to CD45RA+ cells.

Adult↗
PubMed2006

Differential effect of IFN-tau on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers.

IFN-tau is a signaling protein secreted by the bovine conceptus during the peri-implantation period and responsible for pregnancy recognition. Its main role is the prevention of pulsatile release of luteolytic PGF2alpha, but it also exerts immunomodulatory activities characteristic for other type I interferons. The aim of the study was to examine the effect of IFN-tau on the proliferation and distribution of peripheral blood lymphocyte subsets during one-way mixed lymphocyte reaction (MLR) in cows and heifers. IFN-tau inhibited the proliferative response of lymphocytes in MLR both in cows and heifers in a dose-dependent manner, but cow lymphocytes were less susceptible than those ones from heifers. It was also showed that IFN-tau differentially changed lymphocyte subsets distribution in MLR in cows and heifers. In cows, the relative percentage of CD8(+) cells after MRL in the presence of IFN-tau was significantly lower than in heifers. Differential effect of rIFN-tau on proliferation and lymphocyte subsets distribution in a one-way MRL in cows and heifers indicated that the age of the mother is an important factor in immunomodulatory effect towards developing bovine embryo.

Analysis of Variance↗
PubMed1985

Lymphocyte subsets, phytohaemagglutinin responsiveness of blood lymphocytes, and interleukin 2 production in sarcoidosis.

To test the possibility that T lymphocyte subset imbalance and interleukin 2 (IL2) play a part in the impairment of cellular immune response in sarcoidosis, the proportion of T lymphocyte subsets in peripheral blood and alveolar lavage fluid from 21 patients with sarcoidosis was studied, monoclonal antibodies OKT3, OKT4, and OKT8 being used. Lectin induced production of IL2 and phytohaemagglutinin (PHA) responsiveness of peripheral blood lymphocytes were investigated. The percentage of both OKT3+ and OKT4+ T lymphocytes was significantly lower in peripheral blood from patients with sarcoidosis than in control subjects (control 63% and 46%), more so in patients with chronic sarcoidosis (44% and 23%) than in patients with recent sarcoidosis (56% and 38%). PHA induced IL2 production from peripheral blood lymphocytes did not differ between patients with sarcoidosis and control subjects. There was a significant positive correlation between PHA responsiveness and the percentage of blood OKT3+ and OKT4+ cells. Peripheral blood lymphocyte PHA responsiveness was decreased only in patients with an OKT4/OKT8 ratio of less than 1.5. Finally, late addition of exogenous IL2 to the culture medium on day 5 increased 3(H)Tdr incorporation by PHA stimulated blasts in peripheral blood lymphocytes from normal subjects, but not from those of patients with sarcoidosis. The data suggest that the impairment of cellular immune response in patients with sarcoidosis could in part reflect a decrease in the percentage of blood T helper lymphocytes and impaired IL2 receptors at the surface of stimulated lymphocytes.

Adult↗
PubMed1993

Technical influences on immunophenotyping by flow cytometry. The effect of time and temperature of storage on the viability of lymphocyte subsets.

The typing of lymphocyte subsets may be influenced by a variety of technical influences including the duration and temperature of sample storage and the method used for staining samples. We have extended a previous study examining the effect of storage conditions on the baseline values of a number of lymphocyte subsets. EDTA-anticoagulated samples from 13 HIV-1-positive and 15 healthy laboratory controls were analyzed for a number of lymphocyte subsets (CD3+, CD4+/CD3+, and CD8+/CD3+ T cells and CD19+ B cells) (whole blood lysis method, Becton-Dickinson FACScan flow cytometer and reagents) at 0, 24, 48, 72, and 96 h after storage at 4 degrees C, 17 degrees C or 21 degrees C. During storage at both 4 degrees C and 21 degrees C, there were significant changes in baseline values of the majority of lymphocyte subsets and some of these were related to the HIV status of the donor. The optimum temperature for storage in our system appeared to be around 17 degrees C in both our study groups. We have also used propidium iodide in order to discriminate between viable and non-viable cells during flow cytometry of lymphocytes from eight HIV-1-positive and five control subjects. The results show that for both HIV-positive and control samples stored at 4 degrees C, and for control subjects at 21 degrees C, the changes in baseline values of lymphocyte subsets observed were not due to selective loss of particular subsets arising from cell death during storage. However, there was substantial loss of cells from all three subsets in HIV-positive subjects during storage at 21 degrees C, with loss of CD8+ and CD3+ T cells being more significant than loss of CD4+ T cells.

Blood Preservation↗
PubMed1998

Flow cytometric analysis of lymphocyte subsets in migraine patients during and outside of an acute headache attack.

We have conducted flow cytometric studies of two subsets of lymphocyte markers in groups of migraineurs during (n = 12; group B) and outside (n = 10; group C) of a migraine without aura attack (total n = 22; group A), including a group of patients tested in both of these phases (n = 5; group D), and compared these results with those obtained from a population of age-comparable, sex- and race-matched healthy volunteers (n = 12; group E). Comparison of the first set of lymphocytes (CD3+CD16 + 56+, CD3-CD16 + 56+, CD3-CD19+, CD3+CD19+, and CD3+HLA - DR+) between the patients in group A and the controls (group E) showed differences, reflecting greater group A percentages of CD3+CD16 + CD56+ and CD3-CD19+ lymphocytes. Furthermore, these differences reached statistical significance only for the CD3+CD16 + CD56+ lymphocytes, and then solely for the patients in group C (Scheffe's test, p < 0.05). Paired analysis of the above lymphocyte markers for subjects in group D failed to show significant differences between patients when they were having and not having a migraine attack, raising the possibility that results from a larger study could show meaningful increases in percentages of CD3+CD16 + CD56+ lymphocytes as one of the immune parameters useful for differentiating migraineurs from controls. Comparison of a second set of lymphocyte markers (CD19+CD5+, CD20+CD72-, CD20-CD72+, CD20+CD72+) among either the different groups of patients or between the patients and controls failed, however, to show statistically significant differences, emphasizing the apparent specificity of the findings described above for CD3+CD16 + CD56+ lymphocytes. Our results, albeit of a preliminary nature, suggest the occurrence of significant, differential changes in lymphocyte subset immunophenotyping between groups of pain-free migraineurs and patients during an acute migraine episode or controls. Corroboration of these findings may prove useful in clinical laboratory practice to identify changes in immunological parameters specifically associated with migraineurs, and help towards a better understanding of the etiology and pathophysiology of this condition.

Acute Disease↗
PubMed2002

[Study of lymphocyte subsets and its activated-molecules in patients with paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: To investigate the relationship between subsets of lymphocytes and between its activated status and the clinical manifestations in patients with PNH, and to unfold immunological mechanism in the pathogenesis of PNH. METHODS: The peripheral blood mononuclear cells (PBMNC) from 18 PNH patients and 20 controls were separated into two subpopulations using anti-CD(59) monoclonal antibody combined with goat-anti-mouse IgG immunomagnetic beads. CD(3)(+), CD(4)(+) and CD(8)(+) lymphocyte subsets were detected by flow cytometry. In 6 newly diagnosed patients, phenotypes associated with T cell activation such as CD(28)(+)/CD(4)(+) or CD(8)(+) cells, CD(8)(+) CD(38)(+) cells, and HLA-DR(+)/CD(4)(+) or CD(8)(+), and NK (CD(3)(-) CD(16)(+)) cells were detected in the peripheral blood. RESULT: Patients with PNH showed significantly increased CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) ratio as compared with controls (1.22 +/- 0.51 vs 0.86 +/- 0.27, P < 0.05), and the CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) ratio in CD(59)(-) PBMC was higher than that in CD(59)(+) PBMC (2.31 +/- 1.56 vs 0.62 +/- 0.27, P < 0.05). The ratios of CD(4)(+) CD(28)(+)/CD(4)(+) markedly decreased and CD(8)(+)HLA-DR(+)/CD(8)(+) increased. CONCLUSION: Patients with PNH appear to have abnormalities in their lymphocytes. Increased ratios of CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) and HLA-DR(+) CD(8)(+)/CD(8)(+) lymphocytes as well as declined ratio of CD(4)(+) CD(28)(+)/CD(4)(+) lymphocytes might be involved in the pathogenesis of PNH.

ADP-ribosyl Cyclase↗
PubMed2000

Hepatitis C virus (HCV) in lymphocyte subsets and in B lymphocytes expressing rheumatoid factor cross-reacting idiotype in type II mixed cryoglobulinaemia.

The IgMk rheumatoid factors (RF) of type II mixed cryoglobulinaemia (MC) react, in 95% of cases, with MoAbs against the cross-reactive idiotypes (CRI) Cc1 or Lc1 (corresponding to the products of the VH1 and VH4 genes). MC is closely associated with HCV infection, a virus which infects lymphocytes and may replicate in B cells. It has been suggested that HCV may induce clonal selection of B cells producing monoclonal IgMk RF in type II MC. To verify whether HCV is enriched in B cells, and in the subsets expressing Cc1 and Lc1 CRI, we studied peripheral blood lymphocytes from eight patients with MC and HCV RNA-positive sera. Seven patients had RF reacting with anti-Cc1, the other with anti-Lc1 CRI. Total lymphocytes, T cells, B cells, and Cc1+ or Lc1+, Cc1- or Lc1- B cells were purified using MoAb-coated magnetic beads. Lymphocyte subsets were then diluted to give a range of 1 x 106-1 x 103 cells and tested for HCV RNA by reverse transcriptase-polymerase chain reaction. HCV was found exclusively in B cells in seven out of eight patients. In three patients HCV was enriched in the Cc1+ cells. In one of these patients, HCV was found exclusively in Cc1+ cells, with Cc1- cells being HCV-. The data indicate that B cells from type II MC patients are almost constantly infected by HCV. In selected cases, B cell subsets expressing IgMk RF CRI are the prevalent cell type infected by HCV. Our data suggest HCV involvement in B cell dysregulation leading to cryoprecipitable IgMk RF production.

Adult↗
PubMed1994

B- and T-lymphocyte subset numbers in the migrating lymphocyte pool of the rat: the influence of interferon-gamma on its mobilization monitored through blood and lymph.

The subset composition of the migrating lymphocyte pool is largely unknown. In order to determine the number of B, T, CD8+, CD4+ and CD4+ 'naive' (CD45RC+) and 'memory' (CD45RC-) lymphocytes in this pool, the thoracic duct lymph of the rat was drained for 7 days. The effect of lymphocyte depletion on the number of blood lymphocytes was also monitored. In addition, the influence of continuously applied interferon-gamma (IFN-gamma) on the mobilization of the migrating lymphocyte pool was investigated. Within 1 week 2 x 10(9) thoracic duct lymphocytes (TDL) were collected, which represents about 50% of the total lymphocyte pool of an adult rat. Among the migrating lymphocytes an early and a late mobilized population could be differentiated. In the former the CD4+ 'naive' (CD45RC+) T lymphocytes constituted the largest population, whereas in the latter it was the B lymphocytes. Continuous infusion of IFN-gamma did not affect the number of lymphocytes in the blood. In contrast, in the thoracic duct IFN-gamma reduced the appearance of all lymphocyte subsets. However, the pattern of reduction over time differed markedly depending on the population (early or late mobilized) and the phenotype (B- or T-lymphocyte subsets). Thus, the migrating lymphocyte pool of the rat is very heterogeneous regarding its populations and shows complex changes in the mobilization pattern after IFN-gamma stimulation. Future studies should focus on how the size and the composition of the migrating lymphocyte pool is regulated.

Animals↗
PubMed2005

Transcriptional response to ionizing radiation in lymphocyte subsets.

Human lymphocyte subpopulations differ in their cellular responses to ionizing radiation. To shed light on the molecular basis of this effect, we characterized the transcriptional response to 1 Gy X-rays of CD4+ T lymphocytes. Of 18,433 genes tested, 102 were modulated more than 1.5-fold. The majority of the strongly activated genes were p53 targets involved in DNA repair and apoptosis. The expression of three of these genes was further tested by quantitative RT-PCR in lymphocyte subpopulations [CD4+ and CD8+ T, CD19+ B, CD56+ natural killer cells and peripheral blood lymphocytes (PBLs)] from ten adult donors. In contrast to DDB2, TNFRSF10B and BAX were differentially modulated among the subpopulations and the PBLs, being more activated in irradiated CD19+ B and CD8+ T lymphocytes. The level of BAX activation in the various subpopulations correlated with the sensitivity of the cells to radiation, suggesting its possible role in the differential radiosensitivity of hematopoietic cell subsets.

Adult↗
PubMed1983

Human natural killer cells analyzed by B73.1, a monoclonal antibody blocking Fc receptor functions. I. Characterization of the lymphocyte subset reactive with B73.1.

We describe the production of the monoclonal antibody B73.1, reacting with a subset of human lymphocytes and, in about one-half of the donors, with neutrophilic polymorphonuclear leukocytes. In the peripheral blood from normal adult donors, 14.6 +/- 8.5% of the lymphocytes react with B73.1 antibody. The B73.1(+) lymphocyte subset does not bear markers of typical T or B cells and corresponds to the lymphocyte subset containing antibody-dependent killer (K) and natural killer (NK) cells. We demonstrate that: a) virtually all lymphocytes with K/NK cytotoxic activity are found in the lymphocyte subpopulation bearing the B73.1-defined antigen; b) the B73.1(+) lymphocyte subset bears the combination of antigens known to be present on K/NK cells; and c) there is a positive correlation between the level of cytotoxicity and the actual number of B73.1(+) lymphocytes in individual donors. We also report the distribution of B73.1(+) lymphocytes according to donor age and tissue types. The use of the B73.1 antibody in quantitating the actual number of K/NK cells and in performing functional studies on spontaneous cytotoxicity is discussed.

Adolescent↗
PubMed1991

Stressor-induced changes in mitogenic activity are not associated with decreased interleukin 2 production or changes in lymphocyte subsets.

Splenic lymphocytes from Lewis rats that received presentations of physically aversive electric shock demonstrated a marked reduction in responsiveness to T-cell mitogens such as concanavalin A. This study examined cellular mechanisms which may be responsible for this functional alteration. There was no difference in distribution of T-cell subsets from shocked and nonshocked rats. There was no difference in the production of interleukin 2 (IL-2) nor was there a difference in the percentage of IL-2 receptor positive T cells or T-cell subsets after culture for 24 hr. However, there was a marked lack of mitogenic stimulation in splenocytes from shocked rats when stimulated with the calcium ionophore A23187. This indicates a defect in the biochemical pathways necessary to activate T-cell mitogenesis.

Animals↗
PubMed1994

Effects of acute exercise on lymphocyte subsets and metabolic activity.

Lymphocyte subsets, their responsiveness to mitogen and their capacity for glutamine oxidation and glycolysis were assessed in seven subjects before and after an acute bout of interval exercise, the purpose being to establish whether exercise is associated with alterations in lymphocyte metabolic capacities. The subjects exercised at 112% of their maximal work capacity (as determined by pre-test) on a treadmill and performed 25 repeat tests, each of 1 min duration interrupted by 2 min rest periods. Venous blood samples were taken at rest and 3 min following completion of exercise. Acute exercise was associated with significant decreases in the percentage of T- (p < 0.01) and B-cells (p < 0.01) and an increase in the percentage of NK-cells (p < 0.05). These changes were accompanied by a significant decrease in the responsiveness of peripheral blood lymphocytes to the mitogen concanavalin A (p < 0.05). Acute exercise was also associated with profound changes in the metabolic capacities of peripheral blood lymphocytes: rates of 14CO2 production from [U-14C]glutamine (19%: p < 0.05) and lactate (27%: p < 0.05) production were increased significantly in response to interval exercise. Linear regression analysis revealed significant correlation between the exercise-mediated changes (%) in T- and NK-cells and changes (%) in both lymphocyte responsiveness to concanavalin A and metabolic capacity, particularly glutamine oxidation to CO2. One interpretation of these data is that acute exercise promotes a redistribution in lymphocyte subsets, and that it is this redistribution that is the basis of both the impairment in lymphocyte responsiveness to mitogens and the increase in lymphocyte metabolic capacity, especially glutamine oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Effect of dietary high doses of vitamin E on the cell size of T and B lymphocyte subsets in young and old CBA mice.

Using anti-CD5 and anti-SIgM fluorescent monoclonal antibodies, four subsets of lymphocytes can be distinguished in CBA mice, SIgM+ (T2) and SIgM- (T1) T lymphocytes and, CD5+ (B1) and CD5- (B2) B lymphocytes. L3T4 anti-CD4 and Lyt2 anti-CD8 positivities delineate two major T lymphocyte subsets. The cell size of these subsets has been evaluated by their forward light scatter in flow cytometry after cell fixation. The mean cell size of the different subsets differs, according to subset, age and vitamin E treatment. Globally, there is an age-related increase in size for all subsets. In vitamin-E treated young animals, all subsets are smaller, and the percentages of the biggest B1 and B2 cells decrease. In old mice, the vitamin-E effect is far more variable. B2 cells tend to increase in size but the percentage of the biggest cells diminishes. On the contrary, there is a marked expansion of the large B1 cells. No effect is discernible on CD5+ T lymphocytes, but L3T4 and Lyt2 subpopulations increase in size. This study is a retrospective one and the mechanisms affecting cell size are speculative. Since the lymphocyte cell size was measured after fixation in an hypertonic medium devised for human blood processing, we cannot differentiate a real size modification from a differential volume resistance to experimental conditions. Whatever the case, the changes in cell volume argue for age-related changes in cell membrane permeability and volume homeostasis. For some subsets, cell activation and consequent size increase must also be considered. As far as vitamin E has marked anti-oxidant properties, its effect on cell size provides indirect evidence for a role of free radicals in the observed changes and gives support to the oxidant stress theory of ageing in immune senescence.

Aging↗
PubMed1987

Histological analysis of lymphocyte subsets infiltrated into mouse tumor tissue exposed to local irradiation.

The subsets of lymphocytes infiltrated into mouse tumor tissue exposed to irradiation were analyzed by the avidin-biotin-peroxidase procedure using monoclonal antibodies. The effect of PSK administration was also analyzed. Microscopic observation revealed the intense lymphocytic reactions in the exposed tumor, especially 7 days after irradiation of 20 Gy, which mainly consisted of anti-L3T4 positive cells, whereas the degree of infiltration was lower in the nonexposed tumor. PSK was found to enhance T lymphocyte subset infiltration into the exposed tumor tissue.

Animals↗
PubMed1996

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in normal pregnancy.

PROBLEM: In normal pregnancy the maternal immune system should be directed towards tolerance or suppression in order not to reject the partly foreign feto-placental unit. The aim of this investigation was to find hallmarks of systemic immunosuppression during normal pregnancy. METHODS: Five healthy primigravidae were examined during pregnancy and postpartum with flow cytometric analysis to define T and B lymphocyte subsets in peripheral blood. In addition, we studied the proliferative response of lymphocytes to mitogens or interleukin-2 (IL-2) alone or in combination with immunomodulating drugs or interleukin-4 (IL-4). The results were compared to healthy, non-pregnant women. RESULTS: During pregnancy and early puerperium we noted an immune balance in favour of suppression, as measured by increased numbers of T "helper/suppressor" (CD4+CD45RA+) and "suppressor"/effector T cells (CD8+S6F1-), and decreased numbers of T "helper/inducer" (CD4+CD29+), T "helper/memory" (CD4+CD45RO+), killer/effector T cells (CD8+S6F1+), and Natural Killer cells (CD56+), as well as decreased numbers of activated lymphocytes expressing IL-2 receptor (CD25+) and T cells expressing HLA-DR (HLA-DR+CD3+). During pregnancy, lymphocyte proliferation was impaired in autologous serum with concanavalin A (ConA), phytohemagglutinin (PHA), or IL-2. A difference in proliferative response to PHA or IL-2 between cultures with AB serum and autologous serum is suggestive of an immunosuppressor factor in serum during pregnancy. Indomethacin significantly increased lymphocyte proliferation in autologous serum with ConA, indicating PGE2 mediated suppressor activity during pregnancy. Chlorambucil and cimetidine modulated the proliferative response to ConA, indicating an alkylating agent sensitive and a histamine dependent suppressor activity during pregnancy. CONCLUSIONS: During normal pregnancy, a state of systemic suppression of the maternal immune system seems to be present.

Adult↗