Ikaros promotes rearrangement of TCR α genes in an Ikaros null thymoma cell line.

Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4(+) CD8(+) thymocyte using an Ikaros null CD4(-) CD8(-) mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR ß gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry nonfunctional rearrangements on both alleles of their TCR α loci. Retroviral reintroduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αßTCR(+) cells. The process is RAG dependent, requires switch/sucrose nonfermentable chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/nucleosome remodeling and deacetylase complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αßTCR(+) CD4(+) CD8(+) thymocyte transition.

Expansion of CD4+CD28null T-lymphocytes in diabetic patients: exploring new pathogenetic mechanisms of increased cardiovascular risk in diabetes mellitus.

AIMS: Diabetes mellitus (DM) is associated with high incidence of first and recurrent cardiovascular events, especially acute coronary syndromes (ACSs); however, the mechanisms involved are still unknown. We sought to investigate the role of CD4(+)CD28(null)T-lymphocytes, a rare long-lived subset of T-lymphocytes with proatherogenic and plaque-destabilizing properties, in the increased cardiovascular risk associated with DM. METHODS AND RESULTS: CD4(+)CD28(null)T-cell frequency was analysed by flow-cytometry in 60 DM patients without overt cardiovascular disease (cDM), in 166 ACS patients with or without DM (ACS/DM+, n= 51 and ACS/DM-, n= 115), and in 60 healthy individuals. The incidence of cardiovascular events (death, myocardial infarction, unstable angina) was assessed at 36 months follow-up. CD4+CD28(null)T-cell frequency (median, range) was higher in ACS/DM+ (12.7%, 0.1-48) vs. ACS/DM- (3.9%, 0.2-35), cDM (3.1%, 0.3-22.4), and controls (1.5%, 0.1-9.1) (P< 0.001 for all comparisons). Notably, cDM patients had significantly higher CD4+CD28(null)T-cell frequency than controls (P= 0.001). Glycosylated haemoglobin A(1c) was the only parameter independently associated with CD4+CD28(null)T-cells in cDM. The 36-month event-free survival was significantly lower in cDM patients with CD4+CD28(null)T-cells ≥4% (90th percentile of normal distribution) than in those with CD4+CD28(null)T-cells <4% (P= 0.039). Among ACS patients, the 36-month event-free survival was the lowest in those with DM and CD4+CD28(null)T-cells ≥4% and highest in those without DM and CD4+CD28(null)T-cells <4% (P< 0.001), being intermediate in those with only one of these features. CONCLUSIONS: In DM patients, CD4+CD28(null)T-cells are expanded and are associated with poor glycaemic control; they also correlate with the occurrence of a first cardiovascular event and with a worse outcome after an ACS.

Null cell senescence and its potential significance to the immunobiology of aging.

The null cell compartments of human bone marrow and mouse spleen were arbitrarily divided into three subpopulations based upon the ability of cells to acquire T or B cell membrane markers when incubated with poly A:U or ubiquitin. There was an accumulation of T cell precursors with congenital absence of the thymus. In contrast, T cell precursors were reduced and there was an accumulation of uninduced null cells with old age. These observations suggest that there is an intrinsic defect of null cell differentiation with a drift towards more differentiated precursors in T cell differentiation with aging. This could result in a diminution in the range of responses by their progeny, mature T lymphocytes.

Response of non-T, non-B acute lymphocytic leukemia cells to phorbol ester.

Non-T, Non-B acute lymphocytic leukemia cells were cultured in vitro with or without the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a potential modulator of differentiation. The eight cases studied were representative of non-T, non-B acute lymphocytic leukemia (ALL) cells and expressed amounts of la antigens varying from 0.9 X 10(5) to 7.1 X 10(5) molecules/cell; these levels were measured in a cellular radioimmunoassay with 21w4 monoclonal antibody directed at a monomorphic human la determinant. With all cases, TPA caused a significant increase in the level of la. Cultures with TPA expressed 4.3 times the amount of la found on fresh ALL cells, and a correlation was observed (r = 0.92) between the level of la following culture with TPA and that found on fresh ALL cells. A 25% increase in the modal volume of ALL cells was also caused by TPA. There was no detectable induction of surface or cytoplasmic immunoglobulin and no change in the expression of the common ALL antigen. Inhibition of [3H]thymidine incorporation and stimulation of 14C-labeled amino acid incorporation were observed in the presence of TPA, suggesting that the increase in la level occurs concurrently with an increase in protein synthesis induced by phorbol ester. Following culture with TPA, a substantial increase in the ability of the ALL cells to stimulate in a mixed-lymphocyte reaction was obtained. These results suggest that ALL cells, like other cell types, are susceptible to the effects of TPA and respond by changes in cell volume, surface antigen expression, and mixed-lymphocyte reaction stimulating capacity.

[Electrokinetic and functional characteristics of natural killers in mice]. / Elektrokineticheskaia i funktsional'naia kharakteristiki estestvennykh killerov u myshei.

Using adsorption methods and elimination of Thy-1+ lymphocytes, the null cell fraction was obtained. The electrophoretic mobilities of unfractionated splenocytes and different fractions of lymphoid cells and null fractions (to which the natural killers belong) were measured with "Parmoquant-2" device. Simultaneously, the natural killer activity of unfractionated splenocytes and null cell fractions was examined.

Subpopulations of mononuclear cells in ageing: expansion of the null cell compartment and decrease in the number of T and B cells in human blood.

Study of the immune system in ageing has yielded conflicting results. These controversies are mainly due to the selection of the subjects studied. We investigated the mononuclear cell subpopulations in the peripheral blood of subjects fulfilling strict admission criteria meant to exclude persons with diseases that influence the immune system. These criteria are described in the SENIEUR protocol devised by a working group in the framework of EURAGE, the Concerted Action Programme on Ageing of the European Community. We compared two groups of volunteers aged 25-34 years, and 75-84 years. Mononuclear cells were investigated by two-wavelength immunofluorescence combined with phase-contrast microscopy. We found a striking increase in the number of 'null' cells (non-T, non-B, non-monocyte) in the blood of the aged persons. The number of T cells was decreased, especially in the suppressor/cytotoxic subset. The number of B cells was slightly, but significantly, decreased; the number of monocytes did not change. The changes in these cell populations may be related to functional changes, and their quantification could be used to monitor attempts to reconstitute the immune defects in ageing. These findings can also serve as reference values in the study of aged persons not fulfilling the SENIEUR criteria, which, in turn, can contribute to the dissection of the influence of disease versus age on the immune system.

Human natural killer cells: correlation of a lytic assay with a visual binding assay.

Different populations of human effector cells were examined for their ability to bind and to lyse K562 target cells as monitored by a fluorochrome-labeled batch system binding assay and a 6-hour chromium release assay respectively. Binding and cytolysis were found to increase, or decrease, concomitantly with null cell purification, and both were abrogated by trypsin treatment of effectors. At 4 degrees C, cytolysis was abolished whereas binding was only decreased. When binding and cytolysis data were correlated, the nylon wool nonadherent cells (i.e., T and null cells) were found to be the most efficient (i.e., fewer bound effectors per target cell lysed) while mononuclear cells were the least efficient with respect to natural killing. These findings support the current hypotheses of spontaneous killer ontogeny and peripheral blood compartmentalization. Furthermore, this study confirms observations made for mononuclear and enriched T plus null cell populations in single conjugate in agarose assays and extends those to the null cell population--a population relatively enriched for large granular lymphocytes.

The null T cell in pig blood is not an NK cell.

Up to 50% of the blood lymphocytes in young pigs are thymus-derived, lack all subset-specific markers and appear immunologically unresponsive, with no known functional role. In an examination of their possible role in natural killing, NK activity was found in unpurified mononuclear cells and in preparations of unselected and nylon non-adherent lymphocytes (T cells and Null cells). However, NK activity was abolished by removing the E rosette forming T cells using a rat IgM anti-pig CD2 monoclonal antibody and rabbit complement, but not by control treatments with a non-binding rat IgM monoclonal reagent and complement or with any other reagent alone. Thus the resting Null T cell appears not to play a significant role in natural killing.

An established MRL/Mp-lpr/lpr cell line with null cell properties produces a B cell differentiation factor(s) that promotes anti-single-stranded DNA antibody production in MRL spleen cell culture.

The cell line established from the lymph node cells of an MRL/lpr mouse, was found to have null cell properties in that it lacked Thy-1, Lyt-1 and Lyt-2 as well as sIg, and continued to grow in the absence of exogeneously added lymphokines such as IL-2 and IL-3. Interestingly, this cell line (KML1) or a soluble factor(s) produced by it promoted anti-ssDNA antibody production in cultures of MRL/lpr spleen cells. The factor did not induce cell proliferation. Therefore, it is concluded that the cell line KML1 produced at least a B-cell differentiation factor, but not IL-2 or IL-3 as far as detected with the respective lymphokine-dependent cell lines.

Monocyte-mediated augmentation of human natural cell-mediated cytotoxicity.

Normal human monocytes can significantly and rapidly augment natural cell-mediated cytotoxicity (NCMC) against K562 target cells. Approximately 50% augmentation was observed after direct mixture of monocytes with autologous null cells in the 4-hr chromium-release assay. This effect was dependent on the number of monocytes, and B cells and granulocytes were not effective. Coculture of null cells with monocytes and subsequent recovery of null cells for use as effector cells also produced significantly elevated cytolytic activity. This effect was dependent upon the number of monocytes, the length of time of coculture, and the cell donor. Augmentation of NK activity was rapid and observed after 0.5-12 hr of coculture, but suppression was observed after 36 hr; augmentation was observed with high monocyte:null cell (1:1, 1:2) ratios, and no effect was generally observed with lower ratios (1:8). At the single-cell level, the augmentation was associated with an increase in the proportion of target-binding cells which were lytically active. The augmentation of NK activity by monocytes required close cellular proximity, was mediated by a factor which was active or induced only in close proximity of the effector and producer cells, and/or was mediated by a soluble factor with a molecular weight greater than 50,000. This new demonstration that monocytes can augment as well as suppress NCMC may represent another avenue by which NK cell activity may be modulated in vivo.

Surface receptors and immune activity of purified human circulating mononuclear cells. V. Circulating null cells and a factor secreted by the null cells inhibit the synthesis of receptors for Fc mu by T cells in culture.

Receptors for Fc mu (Fc mu R) were not detected on freshly isolated unfractionated circulating human mononuclear cells (MNC) or purified T cells. However, a significant percentage of the T cells (20% to 40%), but not of the unfractionated MNC, exhibited Fc mu receptors following culture for 24 h at 37 degrees C in medium enriched with fetal calf serum. These receptors were newly synthesized by the T cells since they were not detected on T cells cultured for 24 h with cycloheximide in a non-cytotoxic concentration. The absence of Fc mu R on the cultured unfractionated MNC (of which 70% are T cells) suggested that non-T cells within the MNC-B cells and/or null cells and/or monocytes suppressed the synthesis of Fc mu R by the T cells. These cells were individually assayed for their capacity to suppress Fc mu R synthesis by co-cultured T cells. The null cells were able to totally suppress Fc mu R synthesis whereas the B cells and monocytes exhibited no suppressive activity. Null cells cultured for 24 h at 37 degrees C secreted a factor which inhibited the synthesis of receptors for Fc mu by the cultured T cells. This factor is referred to as receptor synthesis suppressor factor or RSSF. On the basis of these findings, it is concluded that (a) the circulating T cells do not possess Fc mu R; these receptors are synthesized de novo by the T cells in culture, (b) the null cells, but neither the B cells nor the monocytes, suppress the synthesis of the Fc mu R by the T cells and (c) the null cells, but neither the B cells nor the monocytes, secrete a factor, receptor synthesis suppressor factor, which can suppress the synthesis of Fc mu R by the T cells.

Selective migration of 'null' cells towards a thymus factor in vitro.

Mouse fetal liver, nude adult bone-marrow and normal adult bone-marrow cells are depleted of theta+, Ig+ cells by the use of in vitro migration through a porous membrane towards a thymus supernatant attractant. The attracted cells are free in suspension, viable and can be obtained in large numbers through the use of multiple migration wells for in vivo testing.

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. II. The mechanism of null cell participation in immunoglobulin synthesis and secretion by B cells.

Immunoglobulins were synthesized and secreted by human B cells cultured with T cells with receptors for FcM (TM) helper cells, monocytes, null cells and PWM for 7 days. Immunoglobulin synthesis did not take place if the null cells were omitted from the cultures irrespective of the duration of the culture period. Null cells incorporated into the cultures at only 25% of their optimal concentration did not affect immunoglobulin synthesis markedly by the cultured B cells. However, the number of B cells in the culture could not be diluted without an accompanying marked reduction in immunoglobulin synthesis. The B cells synthesized and secreted significant quantities of immunoglobulin even when the null cells were added as late as day 6 of the 7-day culture whereas no or very little immunoglobulin was synthesized if the B cells were not present from the beginning of the 7-day culture. It was demonstrated that cultured null cells do not transform into B cells and do not attain their immunoglobulin-synthesizing function. Furthermore, cultured B cells do not transform into null cells and do not attain their helper function. The null cells can also be distinguished from the B cells on the basis of cell-surface markers, receptors, and blastogenic responsiveness to phytomitogens. It is concluded that (i) the human circulating B cells require the null cells, in addition to the TM cells, monocytes and PWM, in culture in order to synthesize and secrete immunoglobulin; (ii) the null cell signal that stimulates immunoglobulin synthesis and secretion by the B cells is probably the last signal following the TM helper cell, monocyte and PWM signals received by the B cells; and (iii) the null cells and the B cells constitute distinct lineages of cells.

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. III. Null cells secrete a factor(s) (human immunoglobulin synthesis/secretion-facilitating factor) that can replace the null cells in the synthesis of immunoglobulin by cultured B cells.

In the accompanying communication, it was demonstrated that the null cells, the TM cells, monocytes and PWM are all obligatory participants in the synthesis and secretion of immunoglobulins by human B cells in culture. Here we demonstrate that the null cells secrete a factor, referred to as human immunoglobulin synthesis/secretion-facilitating factor (HISFF) that can replace the null cells in the cultures. HISFF is distinct from the known T cell-derived interleukins. HISFF functions in an HLA-unrestricted fashion since it can facilitate the synthesis and secretion of immunoglobulins by allogeneic B cells. The null cells cultured with TM helper cells and PWM required monocytes in the culture in order to secrete HISFF. Furthermore, B cells cultured with TM cells in medium containing HISFF, monocyte-derived factors and PWM nevertheless required monocytes in order to respond to the HISFF signal. Thus, the monocyte plays a pivotal role in the secretion of and response to HISFF. Normal levels of immunoglobulin were synthesized even when HISFF was added to the cultures of B cells, TM cells and monocytes, in the presence of PWM, as late as day 6 of the 7 day culture. We conclude that the null cells participate in immunoglobulin synthesis by the B cells by secreting a soluble mediator, HISFF, capable of replacing the null cells in the culture; and that the HISFF signal is the last signal received by the B cell before it begins to synthesize and secrete immunoglobulins.

A novel VHDJH to JH joining that induces H chain production in an Ig-null immature B cell line.

A novel Ig H chain gene rearrangement, a VHDJH to JH joining, was observed in an Ig-null immature B cell line. The preexisting, nonproductive VHDJH complex was replaced by the productive VHJH complex which was generated by the novel joining between the rearranged VH and a germ line JH gene. This VHDJH to JH joining is thought to be a site-specific recombinational event mediated by a putative recombination signal sequence, CACAGCC-12-base-GCAAGAAAG, embedded in the rearranged VH gene including the N region. This sequence might be a novel recombination signal sequence, which had not yet been reported, for so-called recombinase.

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity.

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells.

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.