Survival and PHA-stimulation of gamma-irradiated human peripheral blood T lymphocyte subpopulations

Human peripheral blood T lymphocyte subpopulations were identified and isolated on the basis of their ability to bind IgG (T-G), IgM (T-M), or neither immunoglobulin class (T-null). Lymphocytes were exposed to 0, 0.5, 1.0, 2.5 or 5.0 Gy of 60Co gamma-rays either as a T-cell suspension or as separated T cell subsets. Survival curves, determined 5 days after irradiation, revealed that each subset has radiosensitive and radioresistant portions, and that the T-G cell is the most sensitive subset. Mitotic indices of 48-h cultures showed that the response of unirradiated T lymphocytes to PHA varied greatly among the subsets, the highest indices being obtained for the T-M and the lowest for the T-G cells. With the possible exception of the T-G cells, the subsets are relatively resistant to mitotic effects of gamma-rays. T-G cells suppress the PHA-induced mitotic response of the other T lymphocyte subsets, and this suppressor effect is radiosensitive, being abolished by 1.0 Gy. It is concluded that lymphocytes exposed to greater than or equal to 1 Gy of gamma-rays will have very few dividing B lymphocytes or T-G cells. This together with radiation-induced loss of T-G suppressor action means that the predominant lymphocyte types in mitosis after greater than or equal to 1 Gy are the radioresistant T-M and T-null cells.     

Adhesion molecules on peripheral blood lymphocyte subpopulations in the elderly

Adhesion molecules, such as CD49d, CD50 and CD62L, have important roles in many adhesive interactions involving cells of the immune system. Since it has been shown that many immunological alterations are present in aged subjects, we studied, by means of triple colour whole blood immunostaining and multiparametric flow cytometry, the expression and intensity level (MFI) of these molecules on peripheral blood lymphocyte subpopulations from 23 healthy elderly subjects and 13 young controls. In the elderly a decrease in total peripheral blood lymphocytes bearing CD62L antigen was observed (39 +/- 13% vs 63 +/- 6% and 745 +/- 312/mm3 vs 1,393 +/- 407/mm3; p<0.001), whereas the numbers of lymphocytes expressing CD49d and CD50 antigens were comparable in aged and young subjects. In addition, CD50 and CD62L MFI values on total peripheral blood lymphocytes were higher in elderly than in young subjects (5.23 +/- 1.03 vs 4.18 +/- 0.44, p = 0.001 and 2.60 +/- 0.35 vs 2.21 +/- 0.40, p = 0.005 respectively) while the intensity expression of CD49d was unchanged. The percentages and absolute numbers of T and B lymphocytes expressing CD62L were decreased in elderly compared to young subjects (CD62L+CD3+: 43 +/- 15% vs 66 +/- 9% and 581 +/- 257/mm3 vs 1,028 +/- 418/mm3, p<0.001; CD62L+CD19+: 78 +/- 12% vs 90 +/- 4%, p < 0.005 and 103 +/- 64/mm3 vs 207 +/- 98, p < 0.001). A decrease in the proportion of CD62L bearing NK cells was also observed in the elderly (25 +/- 14% vs 46 +/- 24%, p<0.005), although their absolute number was unchanged. No significant differences were detected in the proportion of T, B and NK lymphocytes expressing CD49d and CD50 antigens and only the absolute numbers of B cells expressing these adhesion molecules were lower in elderly (CD49d+CD19+: 121 +/- 71/mm3 and CD50+CD19+: 107 +/- 73/mm3) compared to young donors (CD49d+CD19+: 248 +/- 112/mm3 and CD50+CD19+: 235 +/- 120/mm3, p < 0.001). Moreover, the intensity of adhesion molecule expression was differentially modulated in the elderly depending on the specific lymphocyte cell population considered. The densities of CD49d, CD50 and CD62L antigens on B and NK lymphocytes from the two age groups were not different; on the contrary, T lymphocytes from elderly donors exhibited increased CD49d (1.69 +/- 0.09 vs 1.62 +/- 0.07, p < 0.05), CD50 (4.98 +/- 1.16 vs 3.77 +/- 0.46, p < 0.001) and CD62L (2.26 +/- 0.38 vs 1.99 +/- 0.37, p < 0.05) MFI values compared to young donors.     

Brain-associated-theta antiserum. Differential effects on lymphocyte subpopulations

Subpopulations of lymphocytes were delineated, both in vitro and in vivo, using brain associated-θ antisera. Lymphocytes were depleted from the cortical areas of the thymus and the T-dependent regions of the spleen. No depletion occurred in the medullary area of the thymus or in the deep cortical areas of the lymph node. These histological findings correlated well with the effects of brain associated-θ antisera using an in vitro cytotoxicity assay. Greater concentrations of antisera were required to cause cell death of lymphocytes from lymph node than lymphocytes from either spleen or thymus. These data would suggest that the θ antigen may be either lost or modulated during the process of lymphocyte differentiation.     

Characterization of culture-induced cytotoxicity from human peripheral lymphocyte subpopulations

Cultured human lymphocyte subpopulations can generate cytotoxicity against K562 leukemia target cells under certain conditions. Such cytotoxicity arises during mixed leukocyte culture or in medium containing fetal calf serum (FCS), mitogenic factors, or interleukin 2. We cultured peripheral blood lymphocytes (PBL) in FCS-containing medium after fractionation of these cells on a Percoll discontinuous density gradient. Higher density cell fractions generated culture-induced spontaneous cytotoxicity (CIC) after 2-3 days in culture. CIC was not due to a loss of suppressor cells during fractionation since culture of PBL prior to fractionation yielded the same results. At least some CIC was associated with the differentiation of higher density cells to newly appearing lower density cells during culture. Most CIC required cells with the HNK-1- OKT3- OKM1+ phenotype. Culture-induced cytotoxicity has some similarities to the previously described lymphokine-activated killing but some important differences are also discussed.     

Interrelation of lymphocyte subpopulations in peripheral blood under cervical papillomavirus infection

T(CD3⁺)-, B(CD19⁺)-lymphocytes and their subsets (CD4⁺, CD8⁺, CD3⁺, DR⁺, CD3⁻ DR⁺) in peripheral blood of patients with CIN I, CIN II, CIN III and cancerin situ associated with HPV infection were evaluated. In peripheral blood of women with CIN II, CIN III and cancerin situ the number of T-lymphocytes which expressed CD3⁺ DR⁺ antigen decreased. In patients with CIN I, CIN III and cancerin situ the level of the CD4⁺ cells decreased; the level of the CD8⁺ cells increased. These patients had a lower CD4/CD8 ratio, the number of B cells being standard. The results may have important implications in the prognosis and immunotherapy of HPV infection. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.     

Peripheral blood lymphocyte subpopulations in sarcoidosis.

We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.     

Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes.

On the premise that the differential effects of glucocorticoids on various aspects of the immune response may be mediated by differences in the glucocorticoid receptors in the effector cells, subpopulations of human peripheral blood lymphocytes were examined for these receptors as well as for glucocorticoid responsiveness. Purified T and non-T lymphocytes, when studied by a sensitive whole cell assay technique, contained equivalent amounts of specific glucocorticoid receptor, which, by binding affinity and specificity measurements, were indistinguishable from each other. Furthermore, under in vitro incubation conditions, macromolecular synthesis in both of these cell populations was inhibited by glucocorticoid at concentrations which saturated the receptor sites. It is concluded that the putative differential effects of glucocorticoids on T and non-T lymphocyte-associated functions are probably not mediated by differences in the glucocorticoid receptors in these cell populations.     

Ferritin in normal human peripheral blood T-lymphocyte subpopulations

Six peripheral blood lymphoid fractions (total lymphocytes, non-T, T, Tar (autologous rosette-forming T cells/precursor), T mu (helper), and T gamma (suppressor) lymphocytes) isolated through rosetting procedures were examined for the presence of ferritin by a direct immunofluorescence technique. Although ferritin was present in all lymphoid fractions studied, a significantly higher proportion of ferritin-containing cells were detected in the T-cell fraction than in the non-T-cell fraction, (mean +/- SD = 7.9 +/- 1.6% and 5.0 +/- 1.2%, respectively). T mu- and T gamma-cell fractions showed a twofold increase in the number of ferritin-positive cells (14.1 +/- 1.4% and 15.4 +/- 2.6%, respectively), as compared with Tar (7.0 +/- 0.9%)-and total lymphocyte (6.9 +/- 1.3%)-cell fractions. These results indicate that ferritin is preferentially distributed in T mu and T gamma lymphocytes and may constitute the basis for explaining some of the roles exercised by these cells in the control of other biological systems.     

Alpha-fetoprotein and human lymphocyte subpopulations.

Peripheral blood lymphocytes were incubated with varying concentrations of alpha-fetoprotein and enumerated for total and "active' T lymphocytes, B lymphocytes, and their proliferative responses to phytohemagglutinin. No significant effect was observed on total T or B lymphocyte proportions. However, there was a dose-related increase in proportions of the so called "active" T lymphocytes. The response of human lymphocytes to phytohemagglutinin was markedly depressed. The alteration in the proportion of active T cells and the inhibition of T lymphocyte response to phyto hemagglutinin by alpha-fetoprotein occurred at higher concentrations than are present in amniotic fluid, serum of pregnant women, or serum of adults, but well within the range reached in fetal serum. The immunoregulatory role of alpha-fetoprotein is discussed.     

The Qa-2 antigen on lymphocyte subpopulations. Mixed lymphocyte culture and cell-mediated lympholysis.

Treatments of spleen cells from Qa-2+ strains with Qa-2 antiserum plus complement (C) have revealed that the Qa-2 antigen is present on restricted functional lymphocyte subpopulations. Anti-Qa-2 plus C reduced the mixed lymphocyte culture response and inhibited the generation of cytolytic effector cells. This treatment, however, did not affect cytolytic effector cells once they were generated.     

Morphometric ultrastructural evaluation of human peripheral blood T lymphocyte subpopulations isolated on the basis of their affinity for sheep red blood cells

The present investigation were undertaken to compare on the basis of ultrastructural morphometric analysis human T lymphocyte subfractions forming rosettes with sheep red blood cells (SRBC). The following T lymphocyte subfractions were obtained: ARFC (active rosettes), T1RFC (rosettes after 1 h at 4 degrees C) and T2RFC (rosettes after 2 h at 4 degrees C). Analysis of the mean cell and nuclei volumes of lymphocyte subfractions led to the separation of two cell groups differing in volume in each donor: a cell group consisting of large cells (T1RFC) and group of small cells (ARFC and T2RFC).     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Synthesis of prostaglandins by subpopulations of human peripheral blood monocytes

The ability of monocytes/macrophages to regulate various aspects of immunologic responses may in part depend on their release of soluble substances such as prostaglandins. Using quantitative gas-liquid chromatography/mass spectrometry, prostaglandin E₂ was found to be the major prostaglandin synthesized in culture by human peripheral blood monocytes. Subjecting these cells to discontinuous density gradient fractionation demonstrated significant differences in the synthesis of prostaglandins E₂ and E₁ among the resulting monocyte subpopulations.     

Excretion of DNA by purified human lymphocyte subpopulations.

A large proportion of DNA synthesized in vitro by human lymphocytes stimulated with plant mitogens or specific antigens is selectively excreted from the cells. To determine if DNA excretion differs among various types of lymphocytes, we examined purified human lymphocyte subpopulations for DNA synthesis and excretion in response to stimulation by L-PHA. The relative proportion of newly synthesized DNA that is excreted by unseparated mononuclear cells, macrophage-depleted cells, T, and B lymphocytes is identical despite great differences in the magnitude of their responses. Low levels of both DNA synthesis and excretion by macrophage-depleted cells and B cells can be increased by reconstitution with macrophages and T cells, respectively. These data indicate that DNA exretion is a general property of lymphocytes stimulated to undergo DNA synthesis by plant mitogens.     

Differential spontaneous killing of human and murine tumour cell lines by leucocyte subpopulations from carp peripheral blood leucocytes

Cell populations from carp (Cyprinus carpio L.) peripheral blood leucocytes (PBLs) were examined for nonspecific cytotoxicities. By using monoclonal antibodies (MAbs) against carp thrombocytes (TCL-HB8) and both neutrophils and monocytes (TCL-BE8), PBLs with a density of 1.08 g ml-1 were separated into three fractions: thrombocytes, a mixture of neutrophils and monocytes, and other cells (mainly lymphocytes), and the separated cells were tested for cytotoxic activities against mammalian tumour cell lines (K562, HeLa, P815 and Yac-1 cell). Consequently, the mixture of neutrophils and monocytes exhibited cytolysis against these target cells, whereas the lymphocyte-rich and thrombocyte fractions did not show any cytolysis. To isolate only neutrophils, which do not contain monocytes, the MAb (TCL-BE8) positive cells from PBLs with a density of 1.08-1.09 g ml-1 were separated. Pure isolated neutrophils showed cytotoxic activities against K562 cells, but not P815 cells. Furthermore, analysis of the cytolytic mechanisms indicated that killing of these cells depended on H2O2 or HOCl. These results suggest that both neutrophils and monocytes are effectors for nonspecific cytotoxicity in carp PBLs, and neutrophils may be distinct from monocytes in their reactivity in cytolysis, including target cell selectivity and/or target cell sensitivity, and the cytolytic pathway. In carp, cytotoxicity of target cells can be mediated by several populations of their leucocytes which have cytotoxic capacities with various recognition and cytolytic mechanisms.     

The immunohistochemical expression of metallothionein in inflammatory bowel disease. Correlation with HLA-DR antigen expression,lymphocyte subpopulations and proliferation-associated indices

Metallothionein (MT) expression in intestinal resection specimens from 41 patients with ulcerative colitis (UC) and 10 patients with Crohn's disease (CD ) was immunohistochemically studied by the avidin-biotin (ABC) method. In addition, the possible relationship of its expression with HLA-DR antigen expression, lymphocyte subpopulations and proliferation-associated indices was studied in order to elucidate the role of this molecule in inflammatory bowel disease (IBD). The MT immunoreactivity was recorded by staining and intensity-distribution scores. MT staining varied in and was mainly localized in the cytoplasm, although a combined nuclear/cytoplasmic reactive pattern was also seen in epithelial cells. MT expression was decreased in UC, and CD compared with normal mucosa. No difference in MT expression between UC and CD was noted. In UC, a gradually decreased expression from remission, to resolving and to active phase was observed. An inverse correlation of MT expression with HLA-DR antigen expression was detected (p = 0.018) in the cases of UC. The data suggest that a low level of MT expression in inflammatory bowel disease and particularly in active phase of UC may indicate a decreased endogenous intestinal protection and it may be implicated in the pathogenesis of the disease.     

Differential expression of H-Y antigen on lymphocyte subsets: analysis by flow cytometry

Expression of H-Y antigen in human white blood cells was measured using flow cytometry with monoclonal antibodies. In this system, lymphocytes were stained preferentially in the male, and to a lesser extent in the female. Analysis of the lymphocyte subsets with biotinylated H-Y antibody conjugated with streptavidin-fluorescein isothiocyanate (FITC) and subset-specific antibody conjugated with phycoerythrin derivative (RD1) revealed differential expression of H-Y among the subsets of the male. In samples from eight men, 41.1% +/- 21.7% of B cells (B1) were stained, compared with 20.7% +/- 12.8% of cytotoxic-suppressor T cells (T8) and 5.4% +/- 3.0% of helper-inducer T cells (T4). In samples from seven women, 12.4% +/- 10.9% of B cells were stained, but staining of T cells was negligible.