Lymphocyte subpopulations in peripheral blood of patients with non-Hodgkin lymphoma

Various lymphocyte subpopulations of peripheral blood of 65 patients with aleukaemic non-Hodgkin lymphoma were studied. The clinically active state and remission in both low and high grade malignancy groups were compared. An elevated lymphocyte count was found in the blood of aleukaemic patients, too. Lymphocytosis in low grade malignancies in the clinically active states was found to be higher than in the corresponding group of the high grade cases. The data showed that the most important factor in the increase of total blood lymphocyte count is the increase in the amount of B-lymphocytes. The TA/TT ratio was significantly reduced in all subgroups. The OKT4/OKT8 ratio was normal in all cases.     

Lymphocyte populations in peripheral blood during normal human pregnancy.

The effects of normal pregnancy on peripheral blood lymphocytes were investigated. The frequencies of thymus-derived and bone-marrow-derived lymphocytes in maternal peripheral blood does not vary significantly throughout pregnancy. This observation implies qualitative rather than quantitative alterations in lymphocyte functions to explain the reduced cell-mediated immune responses seen during pregnancy.     

Characterization of mononuclear cell subpopulations in normal fetal peripheral blood

It is now possible to characterize fetal peripheral blood mononuclear cells (FPBMC) from normal fetuses sampled in utero under ultrasound guidance. The surface phenotype of FPBMC from 25 fetuses, between the 20th and the 26th week of gestation, was studied using standard reagents that are known to delineate mononuclear cell subsets in adult peripheral blood. Results were compared with those obtained in neonates (cord blood) and adults. The major subsets of adult PBMC are represented in fetal blood with few qualitative differences: 20% of FPBMC are not recognized, the percentage of T cells is lower with a higher ratio T4/T8, the fraction of cells that express DR molecule is very high, and the distribution of NK antigens is different in fetuses, B cells and monocytes are in equal proportion. This work represents a prerequisite for future functional studies, and provides normal fetal values that will be useful for prenatal diagnosis of congenital immunodeficiency.     

Characterization of human adenoid cells using surface and functional markers for lymphocyte subpopulations.

Adenoid lymphocytes from children undergoing adenoidectomy were compared with blood cells from the same children using techniques for identifying T cells and B cells. A high proportion of adenoid lymphocytes were immunoglobulin positive cells. Of these only a minor fraction carried receptors for the Fc part of IgG. Adenoid B lymphocytes respond poorly if at all to polyclonal B-cell activators, such as LPS or PPD, which show a different reactivity compared to human splenic cells. The response to anti B2-microglobulin was also different; blood cells responded better than adenoid cells. Thus distinct subpopulations of B lymphocytes reside in different lymphoid organs. The adenoid lymphocyte reactivity might reflect their function in the defence mechanism against infections.     

Lymphocyte subpopulations in the blood of newborn infants

Assays of lymphocyte subpopulations and function have been applied to cells from the cord blood of twenty-four infants. The results are compared with those obtained in healthy adults. T cells, assayed by spontaneous rosette formation with sheep red blood cells (E rosettes) were present in lower proportion in cord (53%) than in adult blood (65%). There was a higher proportion of lymphocytes bearing stainable immunoglobulin in cord (32%) than in adult blood (22%). From the blood lymphocyte counts it was calculated that both T and B lymphocytes are present in greater numbers in the newborn infants' blood than in adults. Comparison of DNA synthesis showed that cord blood leucocytes had a higher spontaneous rate, but there were only minor differences in the lymphocyte mitotic response to phytohaemagglutinin (PHA). The response of cord blood lymphocytes was slightly lower to a submaximal stimulus and higher to a maximal stimulus. There was a correlation between the submaximal response and the proportion of E rosetting cells.

The most striking differences between infant and adult blood lymphocytes were in their cytotoxic activity against homologous target cells (Chang cells). Antibody-dependent cytotoxicity (K-cell activity) was readily detected using cord blood leucocytes, though it was lower than that of adult cells. PHA-induced cytotoxicity was very low in all cord blood samples, and in many cases was almost unmeasurable. This dissociation between the two types of cytotoxic activity is consistent with other evidence that they may be mediated by different cell types.

The assays were also applied to blood samples taken from five mothers of tested infants immediately after delivery. While some differences from normal adults were found with the mothers' lymphocytes they did not mirror those of the cord blood samples. This suggests that the pattern found for cord blood lymphocytes is not due to maternal factors crossing the placenta.

     

Two small lymphocyte subpopulations in human peripheral blood. I. Purification and surface marker profiles.

By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form simultaneously. The dominant marker of these E- Fc- cells was surface Ig, and during 4 days of culture this population did not alter its surface markers. Subset 2 was obtained in two ways following rosette centrifugation with AET-treated SRBC and rabbit anti-human Ig-coated autologous RBC. This 'Null cell population' was shown to be highly variable as judged by the surface markers applied after 4 days of culture, and it is suggested that Null cells contain a number of immature lymphoid cells that may acquire their surface marker during culture. It is concluded that the methods described for purification of small subsets of human lymphocytes are effective and easy to perform and might be used to purify cells for functional studies.     

Thyroid autoantibody synthesis by cultures of thyroid and peripheral blood lymphocytes. I. Lymphocyte markers and response to pokeweed mitogen

Thyroid lymphocytes from Graves' and Hashimoto patients have been investigated and compared with lymphocytes from the peripheral blood. Considerably more lymphocytes (20-30 X 10(6)/g) could be isolated from Hashimoto thyroids than from Graves' tissue (1-5 X 10(6)/g) but the cell suspensions extracted from Hashimoto and Graves' glands were similar in terms of cell surface markers and the ability to synthesize immunoglobulin. Thyroid lymphocytes contained a lower proportion of T cells (OKT3+ cells) and in some cases more B cells than the peripheral blood but the ratio of helper to suppressor T cells (OKT4+:OKT8+ cells) was similar to the values obtained for blood lymphocytes. Further, thyroid lymphocytes (unlike blood lymphocytes) synthesized relatively large amounts of microsomal and/or thyroglobulin antibody when cultured in medium only and these levels were significantly decreased by the addition of pokeweed mitogen. The results of this study provide further evidence for the role of the thyroid as a major site of thyroid autoantibody synthesis and emphasize the importance of characterizing the cells infiltrating the gland in autoimmune thyroid disease.     

Lymphocyte surface markers in acute lymphoblastic leukemia of adults.

Leukemic cells from eight adult patients with acute lymphoblastic leukemia (ALL) were examined for the T-lymphocyte (T-cell) marker of sheep erythrocyte (E) receptors and for B-lymphocyte (B-cell) markers of surface immunoglobulins (SIg), complement receptors and FC receptors. Six of these patients' leukemic cells were devoid of both T- and B-cell markers, and therefore were "null" cells. The leukemic lymphoblasts of a 79-year-old patient had all B-cell markers, including monoclonal IgG K and receptors for complement and Fc. In cells from another patient (21 years old), only complement receptors were found. A review of the literature revealed that, similar to childhood ALL, adult cases of ALL were mostly of "null" cell type. All of the T-cell type was found in approximately 20% of patients. ALL of the B-cell type occurred only rarely. The latter cell type appeared to occur mainly in the middle-aged and the elderly.     

Lymphocyte subpopulations, oxidative burst and apoptosis in peripheral blood cells of patients with multiple sclerosis-effect of interferon-beta

At present, the most efficient therapeutical treatment of multiple sclerosis (MS) is achieved by IFN-beta. However, its in vivo effects remain incompletely understood. If applied parenterally, the hydrophobic IFN-beta acts primarily on blood cells with probable selectivity for functionally different lymphocyte subpopulations, monocytes and granulocytes. We have investigated the expression of the activation marker interleukin-2 receptor-alpha (CD25) on CD3+ T cells, CD19+ B cells, foetal-type gamma(delta)+CD3+ T cells and foetal-type CD5+CD19+ B cells of the peripheral blood. In addition, the oxidative burst activity and apoptosis have been determined in mononuclear and polymorphonuclear blood cells, respectively. The study accompanied a phase III trial with IFN-beta1b (BETAFERON, Schering). Two groups of MS patients with relapsing-remitting course of the disease have been investigated at 8 time points (days 0, 5, 15, 31, 60, 90, 180 and 270 after starting therapy): (1) verum group (n = 8) with application of 8 Mill. units IFN-beta1 b every other day, and (2) placebo group (n = 4) with application of placebo for 3 months and therapy as in (1) from day 90 onward. The main results were: (1) Activated T cells decreased until day 180 in the verum group and return thereafter to pre-treatment values, whereas in the placebo group the values remained relatively stable over the whole observation period. (2) Activated B cells increased between days 90 and 270 in both groups, i.e. after verum application in both groups. (3) Foetal-type B cells were more activated than total B and T cells with increase over time in both groups. (4) Foetal-type T cells exerted relatively stable intra-individual levels with generally low CD25 expression, but punctual CD25 peaks in both groups. (5) The spontaneous oxidative burst was higher in lymphocytes, more variable in monocytes and faster increasing in granulocytes in the verum group than in the placebo group. (6) Apoptosis of mononuclear cells and granulocytes showed similar variations in the verum and placebo groups with the exception of a selective increase over time of the proportion of granulocytes undergoing induced apoptosis in the verum group. It is concluded that IFN-beta has the following main effects on the immune system of MS patients: (1) the T cell immunity is systemically and reversibly suppressed, (2) the foetal-type lymphocytes, which are responsible for the first line of defence of infections, are stimulated in the long range, (3) the oxidative burst activity is increased in lymphocytes and granulocytes and instable in monocytes, and (4) the inducibility of apoptosis in granulocytes is increased. Re-examination of the altered blood cell parameters after long-term IFN-beta therapy is warranted.     

Lymphocyte subpopulations during acute and convalescence phases of malaria

Lymphocytes of normal healthy persons were separated from blood by Ficoll-Hypaque gradient centrifugation and iron-magnet application. peripheral blood lymphocytes (PBL) were stained by various dye-labeled monoclonal antibodies. Cells positive for specific surface markers were enumerated by a fluorescence activated cell sorter (FACS) and fluorescence microscope (FM). The results revealed that the percentages of cells positive with one monoclonal antibody counted by these two techniques were similar while the percentages of cells with double staining were higher when counted by FACS than by FM. Lymphocyte subpopulations of 18 patients infected with Plasmodium falciparum during acute and convalescence period were studied. Lymphocytopenia occurred during the acute infection while total white blood cell counts were normal. PBL of the patients were stained with OKT3, OKT4, OKT8, Leu-11 and a combination of Leu-7, Leu-1 monoclonal antibodies. The absolute numbers of all lymphocyte subpopulations were decreased during the acute infection while T8 positive cells were decreased in both percentage and absolute number. Thus T4:T8 ratio (1.7:1) became higher than normal (1.3:1) at this period. During convalescence phase, absolute numbers and percentages of Leu-7+, Leu-1+ and perhaps Leu-7+, Leu-11- cells which had low NK cell activity were significantly higher than during acute illness. The finding might explain why the NK cell activity was low during the convalescence period.     

Lymphocyte populations in peripheral blood in hyperthyroid and euthyroid subjects.

Thymus-derived and non-thymus-derived peripheral blood lymphocytes were examined by means of E- and EAC-rosette tests. The frequency of these lymphocyte populations was the same in hyperthyroid and euthyroid individuals. It was also demonstrated that PHA responsiveness of lymphocytes from hyperthyroid patients was equal to that of lymphocytes from euthyroid controls and did not change after treatment with 131I.     

Lymphocyte surface antigens in the peripheral blood. Detection by flow cytometry. Applications to the immune system in clinical practice

A flow cytometer enables observation of single cells providing morphological characterization of the cell and identification of cell markers. The cells pass a detection point where they are striked by a sharply focused beam. Light scattering by the cells gives informations about size and granularity. Detection of fluorescence emission permits identification of fluorescent tag such as antibodies. Flow cytometric analysis of immunologic phenotyping allows interpretation of weak fluorescence intensity and detection of two antigens on a single cell with dual fluorescence analysis. Evaluation of B and T cells and their subclasses from whole blood preparations, provides informations of immunological status. Immunophenotyping at the clinical level has been used in a variety of human diseases; immunophenotypic changes have been observed in congenital and acquired immunodeficiency syndromes and auto-immune disorders.     

Lymphocyte surface markers in acute rheumatic fever and post-streptococcal acute glomerulonephritis.

Lymphocyte cell-surface markers were examined in forty children with acute rheumatic fever (ARF) and twelve with acute post-streptococal glomerulonephritis (AGN) and compared to thirty-six normal controls of similar age. Cell-surface-marker studies included surface Ig using fluorescein-labelled F(ab)2 anti-F(AB')2, IgG aggregate binding cells, and EAC rosettes. T cells were identified both as 'active' rosettes and total E-binding cells. Proportions and absolute numbers of cells bearing surface Ig and Fc receptors were elevated in subjects with AGN (Pless than0-01-0-5), whereas proportions of cells producing EAC rosettes were diminished. Patients with acute rheumatic carditis or chorea showed a substantial elevation in proportions and numbers of active T-cell rosettes (Pless than0-01). Streptococcal antigen binding cells capable of forming rosettes with autologous cells coated with group A streptococcal membranes were elevated in the acute phase of both rheumatic fever and acute glomerulonephritis(Pless than0-01). The majority of such cells were removed by passage over insolubilized Ig-anti-IgG columns and appeared to be B cells.     

Lymphocyte subpopulations in pyogranulomas of caseous lymphadenitis.

Pyogranulomas of ovine caseous lymphadenitis (CLA) are encapsulated lesions resulting from infections with Corynebacterium pseudotuberculosis, a bacterial pathogen able to grow within macrophages. Immunohistology of CLA lesions showed a band of lymphocytes lining the inside of the collagen capsule in intimate contact with necrotic tissue, the intracapsular lymphocytes being organized into three layers. The innermost layer, immediately adjacent to the central necrotic tissue consisted of a narrow band of MHC class II staining macrophages. Cells staining for CD4, CD8 and gamma delta T cell markers were unevenly distributed throughout the lymphoid layer, tending to be more numerous immediately external to the macrophage layer. The intracapsular lymphoid tissue contained a high proportion of CD8+ lymphocytes (CD4:CD8, 1.5:1) and of gamma delta lymphocytes (CD4:CD8:gamma delta, 1:0.7:0.8). External to the T cell-rich zone and adjacent to the surrounding collagen capsule was a dense band of cells, a proportion of which stained atypically for CD45R and were tentatively identified as B cells. CD8+ and gamma delta+ T cells showed similar distributions and their relative abundance, compared with CD4+ T cells, was a distinguishing feature of the CLA lesion. Staining for factor VIII-related antigen clearly showed endothelial venules throughout the intracapsular lymphoid tissue. The presence of endothelial venules and the organized architecture of the lymphoid tissue teleologically argues that lymphocytes are continually recruited into chronic CLA lesions and play an important role in the ongoing disease process.     

Lymphocyte subpopulations and function in chronic murine toxoplasmosis.

A long-lasting, chronic infection with Toxoplasma gondii was established in C57Bl/6J mice as a model system of slow parasite infection. We quantified all nucleated cells and Thy-1+ and Thy-1- cell subpopulations in thymus, spleen and peripheral and mesenteric lymph nodes throughout the first 8 months of life of mice infected at 8 weeks of age. We found a physiological pattern of change with age in the lymphocyte subpopulations examined; the pattern was distinctive for each lymphoid organ. These normal patterns were altered in infected mice from the beginning of the observation period. The most prominent findings were (a) a relatively early atrophy of the thymus and (b) no increase in Thy-1+ lymphocyte numbers in either spleen or lymph nodes during the first 3 months post-infection, followed by decreasing numbers afterwards. Other findings were a rapid increase in the number of Thy-1- cells in the thymus before the onset of generalized atrophy; accumulation of erythrocytes in the spleen and splenomegaly during the first 3 months post-infection, despite a steady decrease in the number of nucleated cells; and an increase in Thy-1- cells in the peripheral but not in the mesenteric nodes, thereby diluting the Thy-1+ lymphocyte subpopulation.     

Lymphocyte cell subpopulations during acute post-streptococcal glomerulonephritis: cell surface antigens and binding of streptococcal membrane antigens and C-reactive protein.

T lymphocyte surface markers were examined in 23 patients with acute post-streptococcal glomerulonephritis (AGN) in parallel with normal controls and individuals without nephritis who showed evidence of pharyngeal or skin-sore beta-haemolytic streptococcal infection. Numbers of T gamma cells were similar in AGN and normal controls but were significantly lower (P less than 0.05) than those in skin-sore culture-positive streptococcal infection controls. Numbers of T mu cells were similar in AGN and normal controls but were lower (P less than 0.05) than those observed in streptococcal controls. Percentages of T mu cells were similar in AGN and normal controls but were lower (P less than 0.05) than those recorded in streptococcal infection control groups. Proportions of T cells were reduced during AGN (P less than 0.05). Lymphocytes capable of binding type 12 group A streptococcal membranes were increased (30.4%) in patients with AGN as compared to normal controls (4.1%). Subjects with streptococcal infection alone showed elevated but intermediate relative numbers (10.5%) of lymphocytes binding group A membranes. Increased relative numbers of both B and T lymphocytes binding group A streptococcal membranes were present in both AGN and non-nephritogenic streptococcal infection controls.