Comparative migration of B- and T-Lymphocytes in the rat spleen and lymph nodes.

B-lymphocytes were obtained either by thoracic duct cannulation of thymectomized, irradiated rats or by isolation of complement-receptor-bearing lymphocytes from normal rats. They were labeled in vitro with [³H]-leucine and injected iv into syngeneic recipients from which samples of spleen and lymph node were taken at intervals from 15 min to 48 hr after injection. The sites of initial localisation of B- and T-lymphocytes were identical suggesting that the cells migrated into both organs by a common entrance. The two cell types remained closely associated for several hours in the paracortex of lymph nodes and at the periphery of the periarteriolar lymphoid sheath of the spleen. After 1–6 hr, B-cells segregated from T-cells by moving on into the adjacent part of the lymphocyte corona in the follicular area. By 24 hr, B-cells were evenly distributed throughout the corona. A definite minority of B-cells but no T-cells were seen within the germinal centres. In the spleen, T-cells moved into the central area of the periarteriolar sheath before returning to the blood. The immunological significance of the routes of B- and T-cell migration is discussed.     

The three-dimensional structure of human splenic white pulp compartments.

The precise arrangement of B- and T-lymphocytes in the different compartments of the human splenic white pulp is still largely unknown. We therefore performed a 3D reconstruction of 150 serial sections of a representative adult human spleen alternately stained for CD3 and CD20. The results indicate that the T-cell regions of human spleens may be interrupted by B-cell follicles. Therefore, there is no continuous periarteriolar lymphatic T-cell sheath (PALS) around white pulp arterioles. An arteriole may be surrounded by T-lymphocytes at one level, then run across a follicle without any T-cells around, and finally re-enter a T-cell region. T- and B-cell compartments are intricately interdigitated in the human splenic white pulp. CD4(+) T-lymphocytes and the typical fibroblasts of the T-cell region may extend as a thin shell at the follicular surface within the marginal zone. On the other hand, IgD(++) B-cells continue from the follicular outer marginal zone along the surface of the T-cell region. Our findings indicate that the microanatomy of the splenic white pulp differs between humans and rodents. This may have consequences for the immigration of recirculating lymphocytes and for initial interactions among antigen-specific T- and B-lymphocytes.     

T-cells inhibit Friend murine leukemia virus infection of B-cells in vitro

The ability of splenic T-cells to regulate Friend murine leukemia virus replication in lipopolysaccharide-activated target B-cells infected in vitro was investigated. Removal of the T-cell fraction from spleen cells resulted in an 8- to 10-fold enhancement in the number of productively infected cells in the remaining B-cell-enriched fraction, as compared with unseparated spleen cells, and the addition of increasing numbers of purified T-cells to isolated B-cells prior to infection resulted in a directly proportional reduction in the number of B-cells releasing infectious progeny virus. Separation of splenic T-cells into Lyt 2- and Lyt 2+ T-cells before addition to infected B-cell cultures resulted in inhibition of infection only with the Lyt 2- T-cells; Lyt 2+ T-cells did not inhibit infection, even at high 1:1 ratios. Similarly, separation of splenic T-cells into L3T4+ and L3T4- T-cells before addition resulted in inhibition by L3T4+ but not L3T4- T-cells. Also, cytotoxic treatment of splenic T-cells with monoclonal anti-L3T4 antibody and complement before addition to B-cell cultures destroyed the regulatory effects. Finally, depletion of macrophages from both T-cells and B-cells before infection and coculture had no effect on the ability of T-cells to regulate B-cell infection. Collectively these results demonstrate that L3T4+ T-cells can inhibit Friend murine leukemia virus replication in target B-cells. Culture of isolated splenic T-cells with Friend murine leukemia virus in vitro resulted in the induction of alpha/beta but not interferon-gamma synthesis and in some experiments interferon-containing supernatants from T-cell-virus cultures were able to mediate suppression of B-cell infection with Friend helper virus; the addition of antibody specific for interferon-alpha/beta to cultures inhibited the ability of T-cells to regulate B-cell infection.     

Isogeneic and allogeneic lymphocyte interactions may be controlled by cell surface immunoglobulin tropism.

A study of allogeneic MLC (mixed lymphocyte culture) and two types of isogeneic MLC has been conducted in which subsets of B-cells were serologically removed from pools prior to using the remainder as stimulator cells. Cellular division in the two types of ILC (isogeneic lymphocyte culture) was found to be triggered by lymphocytes with IgG₁ on their surfaces. In contrast, the stimulator cells in ALC (allogeneic lymphocyte culture) possessed membrane-bound IgG_2A and/or possibly IgG_2B. Splenic T-cells were incapable of stimulating replication of splenic or lymph nodal T-cells in the absence of B-cells. Splenic T-cell preparations served as weak stimulators of other allogeneic T-cells but only when B-cells, either isogeneic or allogeneic to the stimulator T-cells, were present. We propose that stimulation in the MLC occurs in two distinct steps. First, immunoglobulins on cell surfaces may function to bring appropriate subsets of cells together. Next, antigenic recognition occurs that results in blastogenesis. Furthermore, the tropism or attraction that certain immunoglobulins have for some cell types may determine which subsets of cells participate in allogeneic and which take part in isogeneic MLC.     

DNA polymerase delta mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.     

Cytogenetic and carcinogenic effects in liquidators of Chernobyl accident

A tendency of the increasing frequency of malignant neoplasms in liquidators at low radiation doses was found. The dose-effect dependence of the frequency of radiation markers (dicentric chromosomes) in lymphocytes of peripheral blood was revealed only in a group of liquidators of the ChNPP accident with malignant neoplasms in remote terms.     

Lack of synergistic effect between X-ray and UV irradiation on the frequency of chromosome aberrations in PHA-stimulated human lymphocytes in the G1 stage.

PHA-stimulated human lymphocytes in the G1 stage were irradiated with UV radiation and X-rays, and the cells were analyzed for chromosomal aberrations in the first mitotic division. The frequency of dicentric chromosomes after single X-irradiation in the G1 stage was about twice the yield in the G0 stage. No increase in the yield of dicentrics was observed after combined irradiation with UV and X-rays. This is contrary to the finding for G0 lymphocytes, where a 2-fold increase of chromosome aberrations was observed. UV irradiation of G1 lymphocytes induced chromatid-type aberrations whereas no significant yield of dicentric chromosomes was observed. This is in agreement with previous findings in Chinese hamster cells in the G1 stage [7]. Irradiation of G0 lymphocytes with UV radiation induce a low frequency of dicentric chromosomes. Thus, the present data indicate that the ratio between chromosome-type and chromatid-type aberrations is different in the G1 and G0 stages in human lymphocytes irradiated with UV radiation.     

Quantitative analysis of neutral glycosphingolipids from human lymphocyte subpopulations.

The glycosphingolipids of normal human lymphocytes from individual donors were analysed by high-pressure liquid chromatography. In addition, purified T- and B-lymphocytes were examined separately. Lactosylceramide was shown to be the major neutral glycosphingolipid in human lymphocytes, and monohexosylceramide, trihexosylceramide, globoside and paragloboside were all detected in smaller amounts. Analysis of purified B- and T-cell fractions revealed that each of these populations contained a similar qualitative profile for neutral glycosphingolipids, but that quantitatively, B-cells contained several times more of each glycosphingolipid per cell than did T-cells.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.     

Determination of glutathione in lymphocytes and possible association of redox state and proliferative capacity of lymphocytes.

The glutathione (GSH) content of mouse T- and B-cells was determined and compared with the GSH content of human peripheral blood lymphocytes and human erythrocytes. Owing to the difficulty of obtaining large numbers of purified lymphocytes, a technique was developed to measure picomolar quantities of GSH. By this technique, mouse T- and B-cells, as well as mouse peripheral-blood lymphocytes, were found to contain approx. 30% of the GSH found in human peripheral-blood lymphocytes. The concanavalin A response of human peripheral-blood lymphocytes and human spleen cells was insensitive to 2-mercaptoethanol as well as to culture in 17% O2, whereas mouse lymphocyte responses were altered by 2-mercaptoethanol and inhibited by 17% O2. The capacity of human peripheral-blood lymphocytes, human erythrocytes, mouse T-cells and mouse B-cells to regenerate GSH stores after chemical oxidation by diamide was tested, and it was found that mouse cells were less capable of regenerating GSH than human erythrocytes or human peripheral-blood lymphocytes. In addition, the latter lymphocytes were less sensitive to oxidation of GSH and to inhibition of proliferation by diamide.     

Susceptibility to lipid peroxidation and accumulation of fluorescent products with age is greater in T-cells than B-cells

The age-related loss of immune function, which is primarily due to loss of T-lymphocyte function, is also associated with accumulation in spleen lymphocytes of autofluorescent products indicative of peroxidation damage. In this study, we examined T-cell membranes for age-related changes which could be related to lipid peroxidation. Using fluorescence spectroscopy of CHCl3:CH3OH membrane extracts, we observed that old T-cells have a 2-fold greater accumulation of fluorescent products than old B-cells and that young T-cells, when exposed to free radicals in an in vitro system, accumulate significantly more fluorescent products over time than young B-cells. We used fluorescence polarization to show that young T-cell membranes are more fluid than young B-cell membranes. However, T-cell membrane fluidity decreases with age, whereas B-cell membrane fluidity does not change; in old mice, T-cell membranes are significantly less fluid than old B-cell membranes. Using two-dimensional electrophoresis, we showed that membrane extracts of old T-cells contain many more proteins than extracts of young T-cells. Our results indicate that age-related changes occur in T-cell membranes which could be due to their increased susceptibility to lipid peroxidation and these changes may contribute to the age-related decline in immune function.     

Time course study of the chromosome-type breakage-fusion-bridge cycle in maize.

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. "Healing" of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.     

DNA polymerase alpha does not mediate G0-G1 increase in yield of X-ray-induced exchange aberrations in human peripheral blood lymphocytes

We report experiments to test the hypothesis that the increased yield of dicentric chromosomes observed in human peripheral blood lymphocytes treated with X-rays during the G1 phase of their first cell cycle, as compared with the yield when the cells are treated in their G0 phase prior to phytohemagglutinin stimulation, is a manifestation of the recently-reported conversion of an inactive form of DNA polymerase alpha to its active form as the PHA-stimulated cells pass from G0 into G1 (Sylvia et al., 1988). The specific polymerase alpha inhibitor butylphenyl deoxyguanosine was used as an X-ray post-treatment. The results show that polymerase alpha is not involved.     

Exposure of cynomolgus monkey embryos to retinoic acid causes thymic defects: effects on peripheral lymphoid organ development

We have previously reported that exposure of monkey embryos to 13-cis-retinoic acid (cRA) results in thymic defects. In this study, we analyzed lymphocyte and antigen-presenting cell populations at gestational days (GDs) 80-100 in the thymus, spleen, mesenteric lymph nodes, and gut-associated lymphoid tissue following a teratogenic dosing regimen of cRA (2.5 and 5 mg/kg) at GD14-27. Tissue sections were immunostained for T-cells (anti-CD3), B-cells (anti-CD20), dendritic cells (p55), and major histocompatibility class II (anti-HLA-DR). Digital images of spleen sections were analyzed to obtain the relative area occupied by the cell subsets within the white pulp (WP). Compared with controls, the T-cell dependent compartment of the spleen WP in specimens with perturbed thymic development (aplasia and severe hypoplasia) showed a reduction in size and proportion of CD3(+) T cells. Our findings indicate that cRA-induced thymic defects result in disrupted development of the splenic T-cell dependent compartment.     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

An improved method for preparing G-banded chromosomes from mouse peripheral blood

We describe here several improvements in the method we originally developed to prepare mitotic chromosomes from peripheral blood of laboratory mice. In addition, we have tried several methods to improve metaphase yield from lymphocytes of the inbred strain DBA/2J, which respond poorly to phytohemagglutinin. The yield of mitoses from DBA/2J cells cannot be improved by enhancing T-cell response using interleukin-2 or by using a different T-cell mitogen, concanavalin A. Metaphase yield from peripheral blood cells of DBA/2J mice can be improved significantly by adding lipopolysaccharide to cultures, probably stimulating B-cell as well as T-cell proliferation.     

DNA polymerase δ mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol α and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol α ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Cellular activities during a mixed leucocyte reaction in the teleost sea bass Dicentrarchus labrax

In this investigation a number of "in vitro" activities of sea bass peripheral blood leucocytes (PBL) against allogeneic PBL inactivated by irradiation were studied. Stimulator PBL were cultured with inactivated allogeneic PBL, and direct counting of lymphocytes was done after 2 weeks by immunofluorescence and flow cytometry using mAbs DLT15 and DLIg3 specific for T-cells and B-cells, respectively. In a one-way mixed leucocyte reaction (MLR), results showed an increase of T lymphocytes, whereas B lymphocytes had values similar to those in control PBL. The increase of T-cells in MLR cultures was also confirmed using RT-PCR by analyzing the expression of the T-cell receptor (beta-subunit) mRNA. The addition of 5 microg/ml of cyclosporin A (CsA) to the MLR caused a significant decrease in T-cell proliferation. Leucocytes from MLR cultures displayed an enhanced cytotoxic activity against xenogeneic target cells with respect to control PBL, raising the possibility of the presence of cytotoxic-like T lymphocytes. Cellular activation of PBL was confirmed in 2 weeks MLR by measuring antibody-induced intracellular Ca(++) mobilization with Fura-2 AM. This work represents the first direct quantitative determination of an "in vitro" T-cell activity in a teleost species.