Leu 7+(HNK-l+) Cells

In the present study, combined methods (indirect immunofluorescence with monoclonal antibodies, Percoll density fractionation, FACS analysis, and the cytotoxicity test) were used for further characterization of peripheral blood Leu 7+ cells (human NK and K cells). The Leu 7+ cell content was found to be relatively higher in the low-density cell fraction in which cells of large granular lymphocyte morphology predominated. However, Leu 7+ cells were also present in intermediate and high-density fractions. Low-density Leu 7+ cells were characterized by both Leu 2 (T suppressor/cytotoxic) and OKM1 (myelomonocytic) markers, whereas among high-density Leu 7+ cells the Leu 2 phenotype strictly predominated. Depletion of OKT3+ cells from the non-adherent cell population caused a decrease of cells with T helper and T suppressor phenotypes but did not have this effect on Leu 7+ and OKM1+ cells. After depletion of Leu 7+ cells from the OKT3- population the content of both T suppressor and OKM1+ cells decreased. Both the present results and previous reports enable us to conclude that two main Leu 7+ cell subpopulations are present in blood, namely Leu 7+Leu 2+/Leu 4+ and Leu 7+/OKM1+ cells. The presence of small and large Leu 7+ cells was also shown by FACS analysis.     

Immunohistological phenotyping of thyroid infiltrating lymphocytes in Graves'' disease and Hashimoto''s thyroiditis.

Subsets of lymphocytes in the thyroid were immunophenotyped by their surface antigens in frozen tissues of Hashimoto's thyroiditis and Graves' disease. Using triple layer immunoperoxidase staining (IP), monoclonal antibodies (T3, Leu 3, T8, anti-Tac and Leu 7) were employed to detect markers of T cell subsets, activated T cells, and a natural killer associated antigen. B cells were identified by 2 step IP with anti-IgD antisera. Excluding those cells forming lymphoid follicles, the density of lymphocytes infiltrating between thyroid epithelial cells was much higher in Hashimoto's thyroiditis than in Graves' disease. However, relative proportions of subsets were similar in both diseases. Most of the infiltrating cells were T3 positive T cells (T3+), with more T8+ (suppressor/cytotoxic T) than Leu 3+ (inducer/helper T). Some Leu 7+ were occasionally seen, but surface IgD positive mature B cells (IgD+) were almost absent. In contrast, IgD+ cells were densely aggregated in primary lymphoid follicles and mantle zones of secondary follicles. In these regions, Leu 3+ cells were about twice as frequent as T8+ cells. Some Leu 7+ and scarce Tac+ cells were also found. The present study indicates a major involvement of immunoregulatory T cells in autoimmune thyroid disease, and also suggested intrathyroidal maturation of B cells.     

Interleukin 2 induces appearance of LGL/Leu11+/K562 lytic cells in Leu11- low density peripheral blood mononuclear cell population

Low density Percoll fraction cells cultured with interleukin 2 (IL-2) showed a higher proportion of large granular lymphocytes (LGL) and higher K562 cytolytic activity, as compared to a culture lacking IL-2. Furthermore, in a negatively selected Leu11- population, derived from low density cells, cultured for 7 days in medium supplemented with lymphocyte (L) or recombinant (R) IL-2, there appeared LGL and Leu11+ cells. Moreover, some level of K562 lytic activity and higher proportion of DR+ and Tac+ cells was found as compared to lacking IL-2 culture. Cytofluorograph analysis of cells labelled with propidium iodide revealed that a proportion of the low density Leu11- starting cell population entered the growth cycle while cultured with IL-2. In addition was found that Leu11+ cells evolve during culture with IL-2 into population lacking in part this phenotype marker. The present work shows that precursors of K562 cytolytic cells lacking Leu11 antigen reside in low density cell fraction, and that they may differentiate in LGL/Leu11+ cells.     

Monoclonal antibody analysis of T-lymphocyte subsets in young and aged adults

Eleven monoclonal antibodies identifying surface antigens present on human T lymphocytes were utilized to investigate the effects of advanced age on peripheral blood lymphocyte subsets. Both the proportion and number of lymphocytes recognized by five antibodies reactive with 'pan' T cell antigens (OKT3, OKT11, Leu4, T101 and Lyt3) decreased with age. The percentage of helper/inducer (OKT4+, Leu3a+) cells remained constant; however the proportion of Leu2a+, suppressor/cytotoxic cells declined. There was no change with age in the percent representation of OKT9+ or OKT10+ cells, nor in the ratio of helper/inducer to suppressor/cytotoxic cells (OKT4+/OKT8+ or Leu3a+/Leu2a+). Absolute numbers of helper/inducer (OKT4+, Leu3a+), suppressor/cytotoxic (OKT8+, Leu2a+), OKT9+ and OKT10+ cells were lower in elderly individuals as the result of lymphocytes constituting a lower percentage of the peripheral blood white cell population in this age group. While only small differences existed between the lymphocyte populations of young and aged men; aged women, compared to young women, had more dramatic shifts in their T cell populations. Comparison of antibodies putatively identifying similar (the same) functional groups of T cells demonstrated excellent correlation between the percentage of cells enumerated with the antibodies OKT3+: Leu4+ (r = .951), OKT4+: Leu3a+ (r = .914), OKT8+: Leu2a+ (r = .896), and in the ratio of helper/inducer to cytotoxic/suppressor OKT4+/OKT8+: Leu3a+/Leu2a+ (r = .926) cells.     

Natural killer cells in Behçet''s disease.

We studied natural killer (NK) cell activity and numbers in the peripheral blood obtained from patients with Behçet''s disease (BD) in inactive and convalescent stage, and from healthy controls. Ratios of helper/suppressor cells (OKT4/OKT8) were below 1.0 in patients with active stage and were normal in the convalescent stage of BD. A relative increase of OKT8+ cells and at the same time of Leu 7+ cells was obtained in the active and convalescent BD stages. Double marker analysis revealed that the sub-population of cells expressing both the T8+ and the Leu 7+ antigen (T8+/Leu 7+) was increased in patients with active stage, and normal in the convalescent stage. The frequency of cells reactive with Leu 11 monoclonal antibody (active NK cells) was evaluated in patients with BD. Data from peripheral blood showed an increased sub-population of T8+/Leu 7+ double marker cells, and a decreased Leu 11+ cell sub-population in patients with active BD, but the majority of Leu 7+ cells in patients with convalescent stage lacked OKT8 antigen when investigated in a double marker system. A parallel increase of Leu 11+ cells was observed in the convalescent stage. This phenotypic analysis was carried out with the NK in vitro functional evaluation of cell populations from peripheral blood. NK cell activity in the clinically active stage of BD was significantly lower than that of healthy controls and patients in the convalescent stage. The decrease of peripheral blood NK function in patients with active BD may be related to the presence of immature forms of NK cells and/or to the increased percentage of T8+/Leu 7+ cells.     

Response of human T lymphocytes to phytohemagglutinin (PHA) after sequential depletion of monocytes, HLA-DR+, Leu11a+, and Leu7+ cells

The experiments presented in this paper deal with the question of whether there is an absolute requirement for alpha-naphtylacetate esterase (ANAE)-positive monocytes, HLA-DR+, Leu11a+, and/or Leu7+ cells to stimulate human peripheral blood T lymphocytes by phytohemagglutinin (PHA). Purified (p) T lymphocytes containing less than 0.1% ANAE-positive monocytes were isolated from human peripheral blood mononuclear cells (MNC) by sequential removal of carbonyl-iron phagocytic cells and of low-density cells by density gradient centrifugation and isolation of E-rosette-forming cells (E-RFC). These pT-cells were further depleted of HLA-DR+, Leu11a+, and/or Leu7+ cells using monoclonal antibodies and cell sorting. The T lymphocytes were stimulated by PHA in an ultra-micro culture in glass capillaries at a volume of 1 microliter or 2 microliters, containing 1000 cells per culture. With this method, the accessory cell requirement could be studied under limiting cell number conditions. The results show that pT-cells can be stimulated by PHA in the absence of ANAE-positive monocytes. No ANAE-positive monocytes were found in the culture after stimulation, indicating the lack of differentiation into ANAE-positive monocytes from ANAE-negative precursors. A rabbit antiserum against leukocytic pyrogen (LP, also containing anti-IL 1 activity) only reduced but did not abrogate the stimulation of pT-lymphocytes by PHA. Addition of adherent cells resulted in an enhancement or in an inhibition of the response of pT-lymphocytes to PHA, depending on cell concentration and culture time: The lower the number of cultured T lymphocytes and the shorter the culture time, the higher was the enhancing activity by additional adherent cells, and vice versa. Further purification of the pT-cells using monoclonal antibodies and cell sorting led to the finding that depletion of either HLA-DR+, Leu11a+, or Leu7+ from pT-cells only reduced but did not abrogate the stimulation of the pT-cells by PHA. However, in absence of HLA-DR+ and Leu7+ cells, the pT-lymphocytes totally failed to respond to PHA. This abrogation of the response was not observed when pT-cells were depleted of HLA-DR+ and Leu11a+ cells. In addition, T11+/HLA-DR- T lymphocytes isolated from E-RFC by positive selection in a cell sorter also responded to PHA.(ABSTRACT TRUNCATED AT 400 WORDS)     

T cell subsets in patients with rheumatoid arthritis: reduction of cells with Leu 2A phenotype in patients with serum IgG immunocomplexes

The distribution of T cell subsets in the peripheral blood lymphocytes of 50 patients with rheumatoid arthritis (RA) and 28 controls was evaluated using the monoclonal anti-human T cell antibodies, alpha-Leu 1, alpha-Leu 2a and alpha-Leu 3a. A reduced number of cells with Leu 2a phenotype was observed in the group of patients with active RA. When patients were classified according to both disease activity and the presence of IgG or IgM immunocomplexes, a good correlation was observed between reduced Leu 2a+ numbers and the presence of IgG immunocomplexes. Patients without serum IC had normal numbers of Leu 2a T cells, independently of the activity of the disease. The significance of these results to the understanding of the aetiopathogenesis of RA is discussed.     

Human tonsillar IgE biosynthesis in vitro. II. Analysis of T cell regulation with monoclonal antibodies

Phenotypes of T cells regulating human tonsillar IgE biosynthesis in vitro were studied by use of Leu 2a and Leu 3a monoclonal antibodies that recognize T cell subsets. B cells cultured with Leu 3a+-enriched populations (B cells plus T3a) produced significantly more IgE and IgG in the presence of pokeweed mitogen than B cells with the Leu 2a+-enriched populations (B cells plus T2a) (p less than 0.001 for IgE and p less than 0.001 for IgG). No significant differences were observed in IgE and IgG synthesis between the cultures of B cells alone and B cells plus T2a. T2a, but not T3a cells, significantly suppressed IgE synthesis (p less than 0.05 for geometric means and p less than 0.001 for percent suppression) when the cells were added to cultures of B cells plus T3a. Suppression of IgG synthesis was not observed under conditions that suppressed IgE synthesis, suggesting qualitative and quantitative differences in regulation of production of these isotypes. When T2a cells were irradiated, the suppressor activity disappeared. When graded numbers of T3a cells were added to B cells, it was noted that IgE synthesis first increased and then decreased as the numbers of T3a were increased. When the T3a cells were irradiated, IgE biosynthesis was suppressed at lower T/B ratios (less than 1 in four of five experiments) and was enhanced at higher T/B ratios (greater than 1 in all five experiments). Similar results were observed in experiments with OKT4 and OKT8 monoclonal antibodies. It is concluded that phenotypes of helper T cells for IgE synthesis are Leu 3a+ or OKT4+ and that IgE suppressors are predominantly Leu 2a+ or OKT8+ and are radiosensitive, as reported for regulation of other isotypes. However, it is suggested that Leu 3a+ and OKT4+ cells consist of radioresistant and radiosensitive helper cells and, presumably, a minor population of suppressor cells.     

Comparison of the staining of peripheral blood T lymphocytes by various anti-CD8 and HLA-DR monoclonal antibodies

T cell subset analysis with monoclonal antibodies is performed in variety of clinical situations, including the monitoring of transplant recipients. We compared the percentages CD8+ cells in the peripheral blood of ten normal volunteers and 26 renal transplant patients using fluorescein isothiocyanate (FITC)- or phycoerytrin (PE)-conjugated Leu2a and Leu2b monoclonal antibodies (mAb). Furthermore, the percentage of HLA-DR+ cells within this population was determined using two anti-HLA-DR antibodies of differing isotype (designated 203 and 243) and either an FITC or a PE label. For both controls and transplant recipients, the PE-conjugated Leu2a and Leu2b mAbs gave significantly higher percentages of positive cells than the FITC-conjugated antibodies (P less than 0.01). Furthermore, the percentage of Leu2b+ cells was higher than the percentage of Leu2a+ cells, irrespective of the fluorochrome used (P less than 0.01); this was particularly true for samples with less than or equal to 10% Leu2a+ cells. Cell sorting experiments indicated that up to 30% of the Leu2a- population were able to bind OKT8 or Leu2b mAb in blood samples with a low percentage of Leu2a+ cells. The percentage of Leu2+ cells that were stained with anti-HLA-DR mAb 203 or 243 varied considerably, depending on the fluorochrome and the isotype of the antibodies. We conclude that the analysis of peripheral blood T cells with mAbs that apparently have the same specificity may give significantly different results, depending on the patient population, the fluorochrome and the isotype of the antibodies.     

Possible in vivo modulation of Leu 2a expression on suppressor T cells in active multiple sclerosis

Reduced numbers of suppressor T lymphocytes were identified by monoclonal antibodies in the peripheral blood of patients with multiple sclerosis (MS) in acute relapse. In vitro culture of cells from these patients resulted in a significant increase in the number of cells identified with the suppressor T cell marker Leu 2a but not OKT 8. The expression of other T cell antigens (Leu 4 and Leu 3a) remained unchanged. This change in Leu 2a expression did not occur when cells from healthy controls were similarly treated.     

A flow cytofluorometric double staining technique for simultaneous determination of human mononuclear cell surface phenotype and cell cycle phase

A double staining technique for the simultaneous determination by flow cytofluorometry of cell surface phenotype and cell cycle phase is described. Peripheral blood mononuclear cells were stained with fluorescein-conjugated monoclonal antibodies for cell surface phenotype, fixed serially with 2% paraformaldehyde and 71.25% ethanol, and stained with propidium iodide to label cellular DNA. The cells were then analyzed by flow cytofluorometry for both green and red fluorescence. A variety of cells, including T cells and their subsets, B cells, NK cells and monocyte/macrophages, can be identified by this technique with simultaneous determination of cell cycle phase.     

Correlation of natural killer cell function with Leu 7 reactivity in patients with humoral immunodeficiency.

We studied the surface markers Leu 7, Leu 1, Leu 2a and Leu 3a on lymphocytes of 54 healthy blood donors and 19 patients with humoral immunodeficiencies and compared the relative numbers of positive cells with the natural killer cell (NK) activity. In controls, and in 12 out of 19 patients (group A) the percentage of the Leu 7+ cells was positively correlated with NK activity. Seven of 19 patients had increased relative numbers of Leu 7+ cells and low NK activity (group B). In controls and group A patients about 40% of the Leu 7+ cells simultaneously expressed the cell markers Leu 1 and Leu 2a. In group B patients approximately 70% of the Leu 7+ cells carried T cell antigens. Furthermore, a distinct suppressor T cell predominance was noticed and the large granular lymphocytes of these patients showed morphological abnormalities.     

Flow Cytometric Analysis of Lymphocyte Subsets in the Bronchoalveolar Lavage Fluid and Peripheral Blood of Healthy Volunteers

The lymphocyte subsets in the bronchoalveolar lavage fluid (BALF) and the peripheral blood of 25 healthy volunteers were examined by analysis with a fluorescence-activated cell sorter. Comparison of the lymphocyte subsets in the BALF with those of the peripheral blood revealed much higher values for the ratios of each Leu 3a+ (CD4), Leu 3+8-, and Leu 2+15- cells, while the ratios of Leu 1+ (CD5), Leu 2a+ (CD8), Leu 7+, Leu 8+, Leu 10+, Leu 11a+ (CD16), Leu 12+, and Leu 2+15+ cells were low in the BALF. The above results indicate that the lymphocyte subsets in the BALF from healthy individuals are mainly composed of cells with surface phenotypes of helper T cells and cytotoxic T cells with virtual absence of cells carrying suppressor T and NK cell phenotypes, and with low B cell ratio. Therefore, it is assumed that the local immune mechanism of the lung is different from that of the peripheral blood.     

T lymphocytes compartmentalized on the epithelial surface of the lower respiratory tract express the very late activation antigen complex VLA-1

T lymphocytes on the epithelial surface of the lower respiratory tract are thought to represent a relatively compartmentalized population of T cells that exchanges slowly with the blood. Since the lung is chronically burdened with antigens, "resident" T cells likely have a history of past activation. To evaluate this concept, we analyzed resident lung T cells for VLA-1 expression, which is indicative of a history of past stimulation. Lung lavage and blood T cells were evaluated in 13 normal nonsmokers using the monoclonal antibodies Leu4 (pan T cells), Leu3 (helper/inducer T cells), Leu2 (suppressor/cytotoxic T cells), TS2/7 (alpha 1 subunit of VLA-1), and A-1A5 (beta subunit of VLA-1) using immunofluorescence and immunoprecipitation. In contrast to normal blood T cells which did not express VLA-1, lung T cells expressed the 210-kDa alpha 1 and 130-kDa beta subunits of the VLA-1 complex, the same as blood T cells activated in culture for 3 weeks. Two-color immunofluorescence with Leu4 and TS2/7 showed that 19 +/- 6% of the lung T cells were VLA-1+, suggesting that a significant proportion of T lymphocytes on the alveolar epithelial surface are in a separate compartment from the VLA-1 blood cells. In sarcoidosis, a disease characterized by exaggerated numbers of active Leu3+ T cells in the lower respiratory tract, increased numbers of lung Leu3+ T cells expressing VLA-1 were present on the epithelial surface of the lung (P less than 0.05 compared to normals). These observations are consistent with compartmentalized, chronically stimulated T lymphocytes on the alveolar epithelial surface that exchange with the systemic immune system very slowly.     

Lymphoid stroma of Warthin's tumor: phenotypic analogies with gut-associated lymphoid tissue

Lymphocytes in cell suspensions and cryostat sections of Warthin's tumors (WT) have been phenotypically characterized using a battery of monoclonal and polyclonal antisera to lymphoid cell-associated antigens. The present study shows that in the WT lymphoid stroma, B cells are more numerous than T lymphocytes and include a significant percentage of surface immunoglobulin A-bearing cells. Of the T cells, the Leu 3a+ and OKT4+ lymphocytes are present in higher percentages than the OKT8+ ones. Both T-cell subsets present a topographic distribution which is similar to that found in human tonsils and gut tunica propria. Intraepithelial lymphocytes displaying an OKT8+, Leu 3a-, OKT3- phenotype are also present. These data, together with previous findings which have demonstrated that Warthin's tumor epithelium synthesizes IgA secretory piece, suggest that these cells may modulate the organization of the surrounding lymphoid stroma toward that of a mature lymphatic tissue phenotipically resembling gut-associated lymphoid tissue.     

A flow cytometric method for measuring lymphocyte proliferation directly from tissue culture plates using Ki-67 and propidium iodide

A new method for measuring lymphocyte proliferation in response to mitogens and allogeneic cells without using radiolabelling is described. It utilizes flow cytometry and the monoclonal antibody, Ki-67, which detects a nuclear proliferation antigen. The entire test is performed in standard, 96-well tissue culture plates. Stable, clean nuclear suspensions rather than whole cells were used to avoid non-specific staining. The nuclei were stained by the indirect fluorescent method. Simultaneous measurements of DNA content were possible by dual staining with propidium iodide (PI). The percentage of Ki-67-positive nuclei correlated well with [3H]thymidine uptake and morphologic quantitation of blasts. This method avoids use of radioactive material and is less time consuming.     

T-cytotoxic/suppressor cell phenotypes in a group of asymptomatic homosexual men with and without exposure to HTLV-III/LAV

The number of lymphocytes bearing the Leu 2+ or T8+ (suppressor/cytotoxic) phenotype is elevated in asymptomatic homosexual men. By two-color immunofluorescence using paired monoclonal antibodies (alpha-Leu 2 and alpha-Leu 15, alpha-Leu 2 and alpha-Leu 7, alpha-Leu 7 and alpha-Leu 11), we enumerated phenotypic subpopulations that are associated with cytotoxic, suppressor, or natural killer function. Both cytotoxic (Leu 2+15-) and suppressor (bright Leu 2+15+) cell populations are elevated in homosexual men. Homosexual men who have been exposed to human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) have higher numbers of Leu 2+15- and Leu 2+7- cells than homosexual men who have not been exposed. Phenotypic subpopulations (dim Leu 2+ Leu 15+ and Leu 7-11+) that are associated with the most potent natural killer activity (against K562 target cells) were not found to be elevated in homosexual men.     

A new method to improve the discrimination between lymphocytes and contaminating erythrocytes in flow cytometric analytical techniques

A method is described for the removal of contaminating erythrocytes from immunofluorescence labelled lymphocyte preparations and allowing accurate gating on the lymphocyte population during flow cytometric analysis. The technique involves an improved erythrolytic fixative combined with the DNA stain propidium iodide permitting non-nucleated cells (i.e., red blood cells) to be gated out during analysis. The erythrolytic fixative was shown not to be detrimental to the major T cell markers detected using indirect immunofluorescence. This method will improve the analysis of blood lymphocytes and their subsets in mononuclear cell preparations and more particularly in samples of body fluids and tissue washings or extracts which may contain variable and often large numbers of erythrocytes.     

Impairments in functional subsets of T-suppressor (CD8) lymphocytes, monocytes, and natural killer cells among asbestos-exposed workers

Peripheral blood leukocytes from asbestos-exposed workers were analyzed by dual color flow cytometry using monoclonal antibodies that identify developmental (HLA-DR) and functional (Leu 8) subsets of T helper, suppressor lymphocytes, and monocytes. An increase in the number of T suppressor cells was closely associated with a decrease in T lymphocyte functions while numerical defects in activated monocytes (Leu M3+Ia+) and natural killer cells (Leu 7+) were correlated with a depressed Th/Ts ratio. Furthermore, among asbestos-exposed workers with depressed T cell functions we have demonstrated a significantly higher number of the effector Ts (Leu 2+ Leu 8-) subset which regulates both the Th/Ts lymphocyte system as well as B cells and NK cell activities. These findings identified changes in the T suppressor feedback regulatory loop as being responsible for the immunoregulatory imbalance among long-term asbestos workers. In double blind analyses of demographic and radiographic data these phenotypic changes were not correlated with age, smoking history, or duration of exposure but were associated with radiographic evidence of asbestos-associated effects. This correlation established a direct link between asbestos exposure and the subsequent development of immune dysfunction.     

Graves'' disease: in situ localization of lymphoid T cell subpopulations.

An in situ analysis of immunological features in thyroids from 15 Graves' disease patients has been performed. This study included a search for immune complexes visualized near the follicle's basement membranes with fluorescent rabbit antisera to human IgG, IgA, IgM, C1q, C3 and C9. Cellular immunity was investigated on the humoral side by visualization of B cells and plasma cells. Two series of monoclonal antibodies (OKT3, 4 and 8, Leu 1, 2a and 3a) were used to label the infiltrating cells' membrane. These studies demonstrated the prevalence of T cells, a majority of them with OKT8/Leu 2a suppressor/cytotoxic phenotype. No correlation was found between this observation and peripheral blood T cell subsets analysis.