Release of xenotropic type C RNA virus in response to lipopolysaccharide: acitivity of lipid-A portion upon B lymphocytes.

The present studies demonstrate that lipopolysaccharide (LPS) causes the release of endogenous xenotropic type-C RNA virus from BALB/c spleen cells. The evidence suggests that virus release is stimulated by the lipid-A portion of LPS and primarily involves an action of LPS on B lymphocytes. LPS had little or no effect on virus release by T lymphocytes, macrophages, or fibroblasts. These results indicate that the differentiated state of the cell plays an important role in the regulation of endogenous virus release.     

Studies on nucleoli in rosetting T and B lymphocytes of the human peripheral blood

The incidence of various nucleolar types was studied in human rosetting lymphocytes to provide an information on nucleolar types present in T and B lymphocytes of the peripheral blood. The results clearly demonstrate that both T and B lymphocytes of the peripheral blood mostly contain ring shaped nucleoli ("resting nucleoli") and less frequently other nucleolar types such as nucleoli with nucleolonemata or compact nucleoli ("active nucleoli") and micronucleoli ("inactive nucleoli"). Since all known nucleolar types and particularly micronucleoli may be observed in both T and B lymphocytes, nucleoli in these cells cannot indicate the type or origin of these cells but simply the state of the nucleolar RNA synthesis.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.     

Antigen presentation in acquired immunological tolerance

In acquired tolerance, previous exposure to antigen under certain conditions induces specific unresponsiveness instead of specific immunological memory. It has been studied as an approach to the mechanisms of self-tolerance that operate on immunocompetent T and B lymphocytes once they leave their sites of origin in the thymus and the bone marrow. Possible mechanisms involve induction of specific suppressor cells or inactivation of antigen-specific lymphocytes (clonal anergy) as a consequence of abortive antigen presentation, in which the antigen receptor is effectively engaged but certain poorly defined accessory signals the T lymphocytes require are lacking. We propose that small, resting B lymphocytes, which lack these accessory signals, are the inactivating antigen-presenting cells in acquired tolerance to proteins and to the class II transplantation antigens. B lymphocytes, which can use their antigen receptors to gather and process antigens that are present at very low concentrations, may play a role in self-tolerance. In addition, B lymphocytes and T lymphocytes rendered anergic by encounter with self antigens could persist as self-specific suppressor cells to block an autoimmune response of autoreactive clones that had escaped deletion or anergy.     

Low-molecular weight nuclear RNA components in human lymphocytes cultured without or with phytohaemagglutinin

Purified human lymphocytes were cultured without or with phytohaemagglutinin (PHA) in the presence of radioactive RNA precursors. RNA was extracted with phenol at 0°, 40° or 62°C and separated on polyacrylamide gels. RNA extracted with phenol either in presence or absence of the RNAse inhibitor diethylpyrocarbonate showed no sign of degradation when separated on 2.6 or 3% polyacrylamide gels. Ten percent gel profiles of whole cell or nuclear RNA showed a a number of small mol. wt RNA components (K, L, M, N, A, B, C, D, F) apart from tRNA, 5 S RNA and 5.5 S RNA. Profiles of cytoplasmic RNA showed only components K and L apart from tRNA, 5 S RNA and 5.5 S RNA. L, C, D and F have an electrophoretic mobility similar to the corresponding components in various ascites cells, while M, N and B may be unique for human cells.The low-molecular wt nuclear RNA components (snRNA) are found in non-stimulated as well as in PHA-stimulated cells and the relative amounts of the snRNA components are not changed during PHA-induced transformation. It is therefore concluded that the relative amounts of the different snRNA components are not related to the dynamic state of the cell.     

Control of cell cycle entry and progression in mitogen-stimulated human B lymphocytes

Tonsillar B lymphocytes were stimulated to proliferate by the mitogenic combination of phorbol dibutyrate and ionomycin. Progression through the cell cycle was monitored by measurements of cellular DNA and RNA content using flow cytometry. Changes in surface expression of class II MHC antigens and CD20 antigen were also monitored as early parameters of B lymphocyte activation and cell cycle progression. The results showed that about 60% of the population synchronously entered and progressed through the cell cycle. The transition from the resting state, signaled by increased RNA content, occurred about 12 to 24 hr after stimulation; S phase entry occurred at about 36 hr. Small, variable populations of cells appeared to be unresponsive to the stimuli, either because they were “preactivated” before in vitro stimulation or were already dying. The kinetics of appearance and accumulation of several cell cycle regulated/regulatory proteins were followed by immunoblotting. The proliferating cell nuclear antigen (PCNA) cyclin A and p33^cdk2 proteins were either absent or present in very low amounts in resting cells and first became detectable in increased amount beginning at about 24 hr after stimulation; increased p34^cdc2 protein was not detected until about 36 hr. Increased cellular content and phosphorylation of the p110^Rb protein was already obvious by 24 hr after stimulation. The effects of several immunosuppressive agents were examined using purified B cells. Both cyclosporin A and an FK506 analogue were shown to inhibit proliferation of B lymphocytes, at the low doses also inhibitory to T cells. © 1995 Wiley-Liss, Inc.     

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

T cells in inductive and effector compartments of the intestinal mucosal immune system of nonhuman primates differ in lymphokine mRNA expression, lymphokine utilization, and regulatory function.

To define further the basis of T cell function in the inductive and effector limbs of the normal intestinal immune system, the capacity of mucosal lymphocytes to produce and use lymphokines and their effects on regulation of Ig production were determined in normal nonhuman primates. Northern blots of RNA from mitogen-activated lamina propria T cells contained more mRNA for IL-2 and IFN-gamma than did mesenteric lymph node T cells. In comparison with lymphocytes from peripheral sites, there was high expression of IL-4 and IL-5 mRNA in both mesenteric lymph node and lamina propria T cells. In studies of lymphokine utilization, T cells from lamina propria had high IL-2-induced but no IL-4-induced proliferative responses. In contrast, mesenteric lymph node T cells had high IL-4-induced and lower IL-2-induced proliferative responses compared with lamina propria T cells. Lamina propria T cells had higher helper activity in PWM-stimulated cultures and exhibited less inhibition by IL-4 than did mesenteric lymph node T cells. These data and previous studies suggest that T cells in an inductive site such as the mesenteric lymph node are a mixed population containing both "naive" cells with low potential for IFN-gamma and IL-2 production and differentiated cells with high potential for IL-4 and IL-5 production. In contrast, the data suggest that T cells in the effector compartment of the lamina propria are comprised primarily of differentiated "memory" cells that produce high levels of IL-2, IFN-gamma, IL-4, and IL-5, have high helper activity, and have a more limited ability to proliferate in response to lymphokines such as IL-4.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanisms of IL-10 production in human microglia-T cell interaction.

IL-10, a cytokine with important anti-inflammatory properties, is generated within the CNS during neuroinflammation. The mechanism for its production is poorly understood. Since infiltrating lymphocytes come into close proximity with the macrophage-like cells of the CNS, the microglia, we have used an in vitro human microglia-T cell coculture system to address the mechanisms of IL-10 production. We demonstrate that microglia or activated T cells alone secrete negligible amounts of IL-10, but that their coculture results in significant IL-10 production, which was effected by both cell types. IL-10 generation was cell contact dependent, and treatment with anti-CD40, CTLA-4-Fc, or anti-CD23 decreased the IL-10 content in microglia-T cell cocultures. The combination of anti-CD40 and CTLA-4-Fc reduced IL-10 levels to the negligible amounts seen with T cells or microglia in isolation. By also measuring TNF-alpha levels, specificity of cytokine regulation was observed; while anti-CD40 and CTLA-4-Fc reduced IL-10 and TNF-alpha levels, anti-CD23 did not affect TNF-alpha while attenuating IL-10 generation. Anti-very late Ag-4, which decreased TNF-alpha levels, did not affect IL-10. These results implicate the CD40, B7, and CD23 pathways in IL-10 production following microglia-T cell encounter and have relevance to the regulation of an anti-inflammatory response within the CNS.     

Lymph node cell composition and DNA content in their different lymphocyte populations in mice with tumor induced by murine Moloney sarcoma virus]

The ratio of T and B lymphocytes and DNA content in population enriched by these cells were analysed in the lymph nodes of BALB/c mice with progressively growing sarcomas induced by the MSV Moloney and in untreated mice. T lymphocyte enriched fractions have a higher DNA content in comparison to fractions enriched with B lymphocytes. Sarcoma development promotes a decrease in the relative number of macronucleolar lymphocytes and simultaneous decrease of micronucleolar ones in the lymph nodes, particularly in the regional ones.     

Herpesvirus sylvilagus infects both B and T lymphocytes in vivo.

Herpesvirus sylvilagus infection of cottontail rabbits (Sylvilagus floridanus) was studied as a model of herpesvirus-induced lymphoproliferative disorders. Leukocytosis, splenomegaly, proliferation of T cells and virus production by lymphocytes characterized this infectious mononucleosis-like disease. Approximately two copies of circular herpesvirus sylvilagus genomes per cell were detected in spleen cells at 2 weeks postinfection, and circular genomes could still be observed after 4 months. Circular viral genomes were found in both B and T lymphocytes. Small amounts of linear viral DNA (0.1 to 0.3 copies per cell) were also detected in both B and T cells. These results indicated that the virus did not replicate in the majority of lymphocytes in vivo. Herpesvirus sylvilagus infection in cottontail rabbits could be useful as a model for studying the complex virus-host relationships of lymphotropic herpesviruses and perhaps as an animal model for Epstein-Barr virus infection in humans.     

Augmentation of regulatory T cells (CD4+CD25+Foxp3+) correlates with tumor stage in patients with colorectal cancer

Recent evidence suggests that decline of regulatory T cells (Tregs) play a critical role in the prevalence of autoimmune diseases inhibiting the maintenance of peripheral self tolerance, while its augmentation leads to insufficient antitumor response, accompanied with poor prognosis in various malignancies. Increased number of Tregs (CD4+CD25+FoxP3+) were noticed in peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes (TILs) and/or regional lymph nodes lymphocytes (LNLs) of patients with gastrointestinal tumors. The aim of our study was to investigate the correlation between the percentage of Tregs in peripheral blood of patients with colorectal carcinoma, using flow cytometric technique and tumor stages, classified as Dukes' A, B, C or D and by stage of differentiation. Peripheral blood venous samples were obtained from 92 patients with colorectal cancer and from 30 healthy adult volunteers. Statistical analysis: Linear regression equations were generated using a least-squares method and analyzed for differences of covariance. Statistical significance was calculated by Mann Whitney U-test. Our data has shown that 15% patients with colorectal cancer were classified as Dukes' A, 41% were Dukes' B, 35% were Dukes' C and 9% were Dukes' D. 54% patients with CRC were well differentiated, 11% were poorly differentiated, 20 were moderately differentiated, tage, 4% were mucinous carcinoma and rest of 11% were partly good differentiated with mucinous components. The increased percentage of Tregs in colorectal cancer patients correlates with tumor stage. These results indicate a possible involvement of regulatory T cells in disease progression. New strategies using inhibition or depletion of Tregs are necessary to elucidate the complexity of defective tumor immunity.     

Nuclear retention of 18S ribosomal RNA by human myeloma cells

Summary Normal quiescent lymphocytes regulate their ribosome content by selectively degrading newly synthesized 18S ribosomal RNA. Unlike actively dividing HeLa cells, lymphocytes retain 18 S ribosomal RNA in the nucleus after synthesis instead of immediately transporting it to the cytoplasm. Subcellular fractionation of the highly differentiated human neoplastic lymphocyte RPMI-8226 reveals that this cell line also retains 18 S ribosomal RNA in the nucleus, a trait not displayed by the less differentiated human lymphoblastoid cell line RPMI-4265. These observations suggest that neoplastic cells can be phenotypically characterized by their ribosomal RNA processing patterns.Operated by Union Carbide Corporation with the Department of Energy     

Purine salvage pathway enzyme activities in human T-, B-, and null lymphocyte populations

Deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) result in distinctly different immunodeficient states. In order to better understand the roles of these two purine salvage pathway enzymes in normal immunity we have characterized their activities in peripheral blood T-, B-, and null (non-T, non-B) cell populations. We have found that T lymphocytes have significantly higher ADA activity than B or null cells (P < 0.001) only when expressed as a function of cell protein. Contrary to other reports B lymphocytes cannot be distinguished from T lymphocytes on the basis of PNP activity. When the enzyme activities are expressed per cell number null cells have a much higher PNP activity (mean ± SD = 461 ± 174 ng/hr/10⁶ cells) than either T (57 ± 10) or B (93 ± 51) lymphocytes (P < 0.001 and 0.002, respectively). Null cells also have a significantly greater protein content per cell (55.0 ± 9.6 μg/10⁶ cells) than T (13.3 ± 4.6) or B (18.7 ± 13.8) lymphocytes. When expressed as a function of cell number null cells have a higher mean, though not statistically significant, ADA activity than T and B lymphocytes. These results indicate that normal human peripheral blood T, B, and null cells can be distinguished on the basis of their ADA and PNP activities and that the method of expression of the activities is critical. The significantly higher PNP activity and protein content per cell in null lymphocytes is compatible with a less differentiated cell population which may include B- as well as T-cell precursors.     

VH gene expression by nontransformed pre-B cells upon differentiation in vitro

Mice have more than 1000 VH gene segments, and each pre-B cell must choose a single one for rearrangement to encode the V portion of the antibody H chain. Presumably, all or most of the functional VH gene segments must be chosen by the population of B lymphocytes if the organism is to express the diversity that is observed in the immune system. Control of the selection of a VH gene segment for expression is not understood. We have found that the members of the VH gene family closest to the constant genes, the 7183 family, are transcribed in a manner that is specific for the stage of B cell development after pre-B cells derived from spleens of 6- to 8-wk-old nude mice are induced to differentiate in vitro by a mixture of dendritic cells and mitogen-activated T lymphocytes (DC-T). DC-T from spleens and lymph nodes induce transient high levels of synthesis of RNA from the 7183 VH family, whereas DC-T from Peyer's patches of mice of the same age as those from which spleen and lymph node DC-T were prepared did not induce the expression of RNA from that gene family. Spleen and Peyer's patch DC-T induce secretion of similar total amounts of antibody. Therefore, the RNA synthesis from members of at least one VH gene family is specific both for the lymphoid tissue in which B cell differentiation occurs and for the developmental stage of the B lymphoid cells.     

Decreased hormone content of immune cells in children during acute lymphocytic leukemia (ALL) - effect of treatment

Histamine, serotonin and triiodothyronine (T3) content of different circulating lymphocyte subsets of leukemic (acute lymphocytic leukemia, ALL) and non-leukemic (control) children were investigated by multicolor flow cytometry. The hormone contents of the cells were followed from the time of diagnosis till the end of treatment. Each hormone could be detected in every time in the investigated cell types, although the amounts of them changed during the treatment.T lymphocytes: Significantly lower amount of serotonin was found in each T cell subsets (Th, Tc and activated T lymphocytes) of leukemic children compared to the healthy control group at the time of diagnosis and it was permanently low during the maintenance therapy. The decreased amount of serotonin could be demonstrated in Tc and Th cells even at one year after the end of treatment. However, there was no alteration in the histamine and T3 content of T cell subsets in the time of diagnosis, but significant decrease was detected during the maintenance therapy and after treatment.NK cells: The serotonin and T3 contents of NK cells (both NK and NKT subsets) were significantly lower at the time of diagnosis and during the maintenance therapy. Similar decrease was detected in the case of serotonin in B cells. Although there was no difference in the T3 content of B cells at the time of diagnosis, significantly lower amounts could be detected during the therapy compared to the healthy control group. The serotonin concentration remained low for years after the end of treatment, both in B and NK cells. These observations might have diagnostic and prognostic importance.     

Regulation of helper cell activity by specifically adsorbable T lymphocytes.

Mice immunized with soluble proteins such as human serum albumin (HSA) or ovalbumin (OA) develop in their spleens antigen-specific T and B lymphocytes. These populations of lymphocytes can be separated from each other by different means; e.g. treatment with anti-theta-antiserum and complement removes selectively T lymphocytes, whereas passage through glass bead columns coated with mouse immunoglobulin (Ig): anti-Ig complexes creates a relatively pure population of T lymphocytes. During the course of such separation studies it was observed that the helper capacity of HSA (or OA) immune mouse spleen cells after Ig:anti-Ig column passage frequently was higher than expected from the enrichment in theta-positive cells. In addition, after adsorption onto antigen coated Bio-Gel beads this effect was even more pronounced, i.e., and increase in the relative helper capacity of about 3 or 4 times compared with an increase in the content of theta-positive cells from about 30% to 40 to 50% after adsorption. The present results will demonstrate that the increased helper capacity was a specific phenomenon which was regulated by theta-positive cells. The regulatory cells specifically adsorbed onto antigen-coated Bio-Gel beads have not been successfully eluted by EDTA or excess-free antigen so far, and they were still adsorbed after pre-incubation with anti-Ig antibodies under conditions where specific B lymphocyte adsorption was almost prevented.