Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment.

Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.

Dual functions of the Drosophila eyes absent gene in the eye and embryo.

In eyes absent (eya) mutants, eye progenitor cells undergo cell death early in development. Whereas the phenotype of eya1 is limited to the eye, other mutations are lethal. Genetic and molecular analysis reveals that mutations in one region of the gene cause embryonic lethality, whereas mutations throughout the gene cause defects in eye development. Mosaic analysis indicates that the eya requirement is cell autonomous. In eye-specific mutants, expression in the eye disc is lacking while embryonic expression is normal. Both the type I and type II transcripts are expressed in the developing eye, and expression of either can rescue the eye phenotype. These data indicate a specific requirement for eya function in eye progenitor cells that is normally fulfilled by both transcripts.

Multiple roles of the eyes absent gene in Drosophila.

The eyes absent (eya) gene plays an essential role in the events that lead to formation of the Drosophila eye; without expression of eya in retinal progenitor cells, they undergo programmed cell death just prior to the morphogenetic furrow, leading to an eyeless or reduced eye phenotype. The eya gene has recently been found to be highly conserved to humans, defining a new gene family. Insights into the gene's function in the fly, therefore, are likely to be relevant to the role of its homologs in vertebrates. Detailed studies at the subcellular level indicate that the Eya protein is localized to the nucleoplasm, suggesting a role in control of nuclear events. The eya gene shows expression and roles in tissues other than the eye, including subsets of cells of the adult visual system, brain, and ovary, as well as an elaborate expression pattern in the embryo. Various mutations in the eya gene cause loss of ocelli, female sterility, or lethality. Analysis of the embryonic lethal phenotype indicates that mutant alleles show defects in head morphogenesis. These data indicate that eya has critical roles in morphogenesis of a number of tissues in the animal, in addition to its role in early eye formation. Despite multiple roles at multiple stages of development of the fly, both the type I and type II forms of the protein, when expressed ectopically during larval development, can direct eye formation.

Transvection at the eyes absent gene of Drosophila.

The Drosophila eyes absent (eya) gene is required for survival and differentiation of eye progenitor cells. Loss of gene function in the eye results in reduction or absence of the adult compound eye. Certain combinations of eya alleles undergo partial complementation, with dramatic restoration of eye size. This interaction is sensitive to the relative positions of the two alleles in the genome; rearrangements predicted to disrupt pairing of chromosomal homologs in the eya region disrupt complementation. Ten X-ray-induced rearrangements that suppress the interaction obey the same general rules as those that disrupt transvection at the bithorax complex and the decapentaplegic gene. Moreover, like transvection in those cases, the interaction at eya depends on the presence of normal zeste function. The discovery of transvection at eya suggests that transvection interactions of this type may be more prevalent than generally thought.

The eyes absent gene: genetic control of cell survival and differentiation in the developing Drosophila eye.

The eyes absent (eya) gene is required at an early stage in development of the D. melanogaster compound eye. In eya mutants, progenitor cells in the eye disc undergo programmed cell death anterior to the morphogenetic furrow, rather than proceeding into the pathway of retinal differentiation. A low level of cell death normally occurs at this stage, suggesting that eya activity influences the distribution of cells between differentiation and death. Molecular analysis identifies a nuclear protein expressed in progenitor cells prior to differentiation. Transformation with the cDNA prevents progenitor cell death and allows the events that generate the eye to proceed. eya activity is required for the survival of eye progenitor cells at a critical stage in morphogenesis.

Increased number of Leu-2-bearing non-T cells with natural killer activity in chimpanzees.

Peripheral blood lymphocytes from adult and adolescent chimpanzees, as well as adult humans, were studied for phenotypic surface markers by flow cytometry. Lymphocytes from chimpanzees were found to have increased numbers of Leu-1-, Leu-2+ cells as compared to humans. These cells, following preparative electronic cell sorting, were shown to possess natural killer function. Further analysis of this subpopulation indicated that they lacked responsiveness to a number of T-cell mitogens. The differences in lymphocyte subpopulations between chimpanzees and humans can almost be totally accounted for by the Leu-1-, Leu-2+ cells. Phylogenetic disparity between these two species may also be found within this population.

Proliferation of infected lymphoid precursors before Moloney murine leukemia virus-induced T-cell lymphoma.

NFS/N mice inoculated with Moloney murine leukemia virus (M-MuLV) developed T-cell lymphoma after a 10-week latent period. Expression of lymphoid differentiation antigens, appearance of M-MuLV-encoded cell surface antigens, and rates of cellular proliferation were measured in splenic and bone marrow subpopulations during this latent period. At 2 weeks of age, Thy-1-and surface immunoglobulin-negative null cells of spleen and bone marrow expressed M-MuLV antigens whereas T- and B-lymphocytes did not. During the 3d and 4th weeks, the number of splenic null cells increased to six times the number found in uninfected controls. These null cells included the precursors of lymphocytes and hematopoietic cells. For the remainder of the latent period, the percentage of null cells undergoing proliferation was three times greater in the infected mice, while the total number of null cells remained constant. This proliferation was not accompanied by terminal differentiation or emigration of mature cell types from the spleen. Proliferation was substantially delayed in CBA mice, which are resistant to lymphoma induction.

Ly-2+ T cell enlargement and null cell proliferation occur at the onset of splenomegaly and autoantibody production in New Zealand Black mice.

Splenic T and B lymphocyte and null cell populations were analyzed in NZB mice as autoimmune disease developed during the first year of life. In the B lymphocytes, a progressive shift occurred from the surface IgD bright subset to the surface IgM bright subset. There was a slight increase in the ratio of Ly-2- to Ly-2+ T cells. Splenomegaly was not detected until after 40 wk of age and was primarily due to an increase in the number of null cells. This change was accompanied by an increase in the size, as determined by narrow-angle forward light scatter, of the Ly-2+ but not the Ly-2- T cells, an elevation of IgG-containing plasma cells, and the appearance of anti-erythrocyte autoantibody. The splenic B cell subset distribution and the enlargement of the Ly-2+ T cells were reflected in the peripheral blood, whereas the T cell subset ratio was not. The B cell subset alteration did not correlate with any of the other changes observed. Statistical associations were found between the ratios of T cell subsets, the enlargement of the Ly-2+ T cells, and the increased number of null cells, suggesting a linkage among those late changes that immediately precede the development of overt autoimmune disease.

Differentiation of B lymphocytes in sheep. I. Phenotypic analysis of ileal Peyer's patch cells and the demonstration of a precursor population for sIg+ cells in the ileal Peyer's patches.

The ileal Peyer's patches (IPP) of sheep may be a primary lymphoid organ for b cells since they have a number of important features in common with the bursa of Fabricius of chickens. We have examined the surface phenotype of IPP cells. Approximately 90% to 95% of IPP cells are 'low sIgM+'; that is, they have surface IgM, but in much smaller amounts than peripheral B cells, which are 'high sIgM+'. IPP cells with sIgG or sIgA are very rare. Upon exposure to a tumour promotor, phorbol myristate acetate (PMA), in vitro, low sIgM+ cells differentiated into high sIgM+ cells. The amount of Ia-like antigens on the surface also increased after PMA treatment. Approximately 5% of IPP cells bore no identifiable markers. However, these cells could also be induced into high sIgM+ cells upon exposure to PMA; this may indicate the presence of precursors of sIgM+ cells within the IPP. While PNA (peanut agglutinin) binds strongly to the vast majority of IPP cells, it binds very little, if at all, to B cells obtained from the periphery, unless they have been treated with neuraminidase; this suggests that cells in the B lineage retain their PNA receptors, but that these become masked by sialic acid on mature B cells.

Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompanying the appearance of cytolytic and interleukin 2-producing cells.

At the 14th day of gestation, embryonic thymocytes+ are large, functionally incompetent cells with H-2K+ Thy-1+ B14- Ly-2- L3T4- phenotype, some of which express TL antigen. Differentiation of these cells in organ culture is characterized by: 1) appearance of cells expressing Ly-2 and L3T4 molecules, first among the population of large cells, after 2 days of culture; 2) appearance of small H-2K- Thy-1+TL+B14+Ly-2+L3T4+ and H-2K-Thy-1+TL+B14-Ly-2+ L3T4+ cells between days 2 and 4; 3) accumulation of small H-2K- Thy-1+ TL+ B14- Ly-2+ L3T4+ (but not H-2K- Thy-1+ TL+ B14+ Ly-2+ L3T4+) cells until day 5 of culture, and their subsequent gradual disappearance which is paralleled by an increase of the proportion of medium-sized H-2K+ Thy-1+ TL- B14- cells with various Ly-2 L3T4 phenotypes; 4) appearance and subsequent accumulation of cytolytic and IL 2-producing cells between days 4 and 6. Comparison of these results with the data from similar in vivo studies shows that differentiation of organ-cultured thymocytes rather closely follows the in vivo development only during the first week of culture and shows significant deviations thereafter. Precursors of cytolytic cells and cytolytic effector cells as well as IL 2-producing cells are found among both Ly-2+ and Ly-2- populations of thymocytes, indicating that there is no clear association between Ly-2 phenotype and the ability to kill or to secrete IL 2.

Lyt-2- T cell-independent functions of Lyt-2+ cells stimulated with antigen or concanavalin A.

Approximately 30% of cytolytic Lyt-2+ clones from primed mice are able to proliferate autonomously, i.e., independent of IL 2 derived from Lyt-2- cells after antigenic stimulation. H-2K- or -D-restricted induction of Lyt-2+ cells to autonomous proliferation requires Ia+ stimulator cells. A strict correlation was observed between the ability of Lyt-2+ clones to proliferative autonomously and to induce DH. Eventually, the growth of all Lyt-2+ cytolytic clones becomes dependent on exogenous IL 2, and their ability to induce DH is lost. Small Lyt-2+ cells can also be induced in primary cultures by antigen or concanavalin A to proliferate in the absence of exogenous IL 2. The frequency of autonomously proliferating small Lyt-2+ cells is the same as that of small Lyt-2+ cells proliferating in the presence of exogenous IL 2. IL 2 derived from Lyt-2- cells can augment proliferation of Lyt-2+ cells, but is not obligatory.

Characteristics of amplifier T cells involved in the antibody response to the capsular polysaccharide of type III Streptococcus pneumoniae.

Amplifier T cell activity can be transferred by spleen cells harvested 72 hr after priming with type III pneumococcal polysaccharide (SSS-III) and can be abolished by treating the transferred cells with monoclonal anti-Lyt-1, or anti-Thy-1 antibodies in the presence of complement; thus, amplifier cells represent a distinct subpopulation of T cells. Amplifier T cells were found to be sensitive to irradiation but not to treatment with cyclophosphamide. When amplifier cells were transferred to athymic nude (nu/nu) mice, the enhancement obtained was much greater than that produced in thymus-bearing (nu/+) mice; this is presumably due to the lack of suppressor T cell activity in nu/nu mice that enables amplifier T cell activity to be expressed more fully. Amplifier T cells also were found to be present in peripheral blood; these amplifier T cells were Lyt-2- in phenotype. Although the induction and activation of amplifier T cells appear to be antigen-specific, the product made by amplifier T cells may not be antigen specific in its mode of action. Because amplifier T cells can be induced and activated by exposure to immune B cells, specificity is presumably due in whole or in part to the ability of amplifier T cells to recognize the idiotypic determinants of B cell-associated antibody specific for SSS-III.

Characterization of T lymphocyte and monocyte populations in HLA B8/DRw3 normal individuals and in patients with dermatitis herpetiformis.

Normal individuals who possess the HLA B8/DRw3 haplotype as well as patients with dermatitis herpetiformis have been found to have a number of immunologic abnormalities including decreased numbers of E rosette-positive, Fc IgG receptor-bearing lymphocytes (referred to as TG cells), and increased numbers of cells which spontaneously secrete immunoglobulin. HLA B8/DRw3-positive normal individuals also have an increased risk for the development of a number of immunologically mediated diseases. Since many of these findings are suggestive of B-cell hyperreactivity and since TG cells were initially thought to represent a portion of the T suppressor cell network, we have examined the peripheral blood mononuclear (PBM) cell populations of 14 normal HLA B8/DRw3-positive individuals, 14 patients with dermatitis herpetiformis (all of whom were HLA B8/DRw3-positive), and 9 non-HLA B8/DRw3 individuals using flow cytometry and monoclonal antibodies of the OK and Leu series directed against cell surface antigens. Normal HLA B8/DRw3 individuals were found to have a significantly lower percentage of PBM cells that expressed both OKT8 and Leu-2a when compared to normal non-HLA B8/DRw3 individuals (p less than .05 Student's t-test). When the ratio of T helper cells (OKT4 and Leu-3a) to T suppressor cells (OKT8 and Leu-2a) was calculated for each individual studied, normal HLA B8/DRw3 individuals were found to have a significantly elevated ratio (Leu-3a/Leu-2a = 2.41 +/- .16, mean +/- SEM) when compared to non-HLA selected individuals (Leu-3a/Leu-2a = 1.73 +/- .05) (p less than 0.025). In addition, normal HLA B8/DRw3 individuals had decreased numbers of TG cells when compared to normal non-HLA B8/DRw3 individuals (B8/DRw3 = 6.4 +/- .74%, non-B8/DRw3 = 13.2 +/- 1.0%, mean +/- SEM, p less than .01). In order to determine the cell surface marker characteristics of TG cells, purified TG cells from both normal HLA B8/DRw3 individuals and non-HLA B8/DRw3 individuals were studied using the Leu series monoclonal antibodies and OKM1. Good agreement was found in the percentages of cells expressing each cell surface marker between the two groups. In addition, the TG cells were found to be predominately T cells (78% Leu-1-positive), with both T helper cells (40% Leu-3a-positive) and T suppressor cells (30% Leu-2a-positive) present. These results suggest that the suppressor cell activity associated with the TG subset is not due to a depletion of the T helper cell subset, and that the decreased numbers of TG cells in HLA B8/DRw3 individuals is not due to a preferential loss of cells bearing Leu-1, Leu-2a, Leu-3a, or OKM1.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional studies on B-cell hybridomas with B-cell surface antigens. III. Differentiative response to phorbol esters.

Phorbol esters have been shown to induce differentiation of human lymphoid cells into the mature stage. Murine lymphocytes, however, have not been found to be induced the terminal differentiation by these products. In this study, TH2.52, a subclone of B-cell hybridomas between M12.4.1 B lymphoma of BALB/c mice and normal B cells of C57BL/6 (B6) mice was treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and the differentiative effect of TPA was examined. TPA treatment inhibited the spontaneous proliferation of TH2.52 and induced significant IgM secretion by the hybrid. In contrast, M12.4.1 did not develop any IgM secretion when treated with TPA. The differentiative effect of phorbol esters on TH2.52 closely correlated with their tumor-promoting activity. In addition, the differentiative response of TH2.52 to TPA was completely blocked by retinoic acid (RA). Moreover, TH2.52 cells treated with TPA were demonstrated to decrease the expression of Iab, Iad molecules as well as IgM molecules on the cell membrane by analyses of flow microfluorometry (FMF) and quantitative absorption tests. On the other hand, Iad expression of M12.4.1 did not change under the same conditions. The result clearly demonstrates that TH2.52 cells can be induced to differentiate into IgM-secreting cells after treatment with TPA, followed by the decrease in the expression of B-cell surface antigens on the cell membrane.

Actin activity is high in the immunocompetent fraction of thymocytes.

We have shown previously that actin activity of lymphocytes correlates with their migratory abilities. To determine whether any subpopulation of thymocytes would possess the potential for peripheral migration, sheep thymocytes were separated into four fractions by Percoll discontinuous gradient centrifugation and actin activity of each fraction was determined. The fraction enriched for immunocompetent medullary-type cells showed the highest actin activity among thymocytes, but the activity was significantly lower than that of peripheral lymphocytes. The implications of the observation in relation to thymocyte migration is discussed.

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens.

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.

Functional studies on B cell hybridomas with B cell surface antigens. II. Ia molecules on the cell membrane and the differentiative response to anti-mu antibody.

TH 2.52, a subline of the B cell hybridoma with Iab, Iad, and IgM molecules on the cell membrane, was treated with F(ab')2 fragments of purified goat anti-mouse mu antibody (anti-mu), and the change in the expression of surface Ia molecules was determined by microcytotoxicity assays, quantitative absorption tests, and analyses of flow microfluorometery (FMF). We have previously reported that TH 2.52 cells can markedly generate IgM after stimulation with anti-mu without T cell factors. In the present studies, it was shown that the expression of surface Iab molecules on TH 2.52, originated from normal B cells of C57BL/6 (B6) mice, significantly decreased after treatment with anti-mu. In contrast, Iad molecules derived from M12.4.1 lymphomas did not change under the same conditions. These results indicate that cross-linking of anti-mu with surface IgM molecules on TH 2.52 provides signals for differentiation into IgM-secreting cells; this is followed by a decrease in the expression of Iab molecules on the cell membrane. Furthermore, monoclonal anti-Ia antibody (anti-Ia) did not inhibit the generation of IgM-secreting cells by TH 2.52 cells treated with anti-mu. In addition, la- sublines of TH 2.52 obtained after mutagenesis with ethyl methanesulfonate (EMS), as well as the original TH2.52, could differentiate into IgM-secreting cells in the presence of anti-mu. These findings suggest very strongly that la molecules on the cell membrane are not required for the induction of IgM secretion by B cells treated with anti-mu.

Characterization of T-cell-soluble factors modulating the expression of Ia and H-2 antigens on BALB/c B lymphoma cell lines.

The effect of supernatants of concanavalin A-activated spleen cells (CAS) on the expression of various antigens, especially Ia antigens, on BALB/c B lymphoid cells, was examined. This study demonstrates the following: (i) CAS enhanced the expression of Ia antigens on four out of five BALB/c lymphoid cell lines. (ii) CAS selectively modulates the expression of Ia and H-2D, but not sIgM or viral gp70 expression, on X16C 8.5 tumor cells. The enhanced levels of Ia expression on B lymphoid tumor cells were also detected by using anti-Ia monoclonal antibodies. (iii) The molecular weight of soluble factor(s) affecting Ia and H-2 was approximately 40,000 estimated by gel filtration on a Sephadex G-200 column. (iv) Type 1 interferon but not interleukin 1, interleukin 2, or T-cell-replacing factor enhanced the expressions of Ia and H-2D antigens. (v) The activity of CAS-modulating Ia and H-2 antigens was eliminated by acidic treatment. It was concluded from this study that at least one of the factor(s) in CAS, modulating the antigenic expression of B-lymphoid cells, was interferon-like in nature. From our findings, a possible immunoregulatory mechanism by interferon was suggested: T cells, after stimulation of mitogens or antigens, secrete interferons which modulate the expression of Ia and H-2 on B cells. Then B cells, whose Ia and H-2 were modulated selectively by T-soluble factors(s), might interact with T cells much more efficiently.

Isolation of human basophils by flow microfluorometry.

Human mononuclear cells labeled with fluorescein-conjugated anti-IgE antibody were subjected to flow microfluorometric analysis. Basophils were enriched to 97-99% purity with a 2-step cell sorting procedure. Trypan blue exclusion of sorted cells exceeded 90% and the net yield of the procedure was 15%.