cAMP potentiates H(2)O(2)-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation.

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.

Regulation of the calcium/NF-AT T cell activation pathway by the D2 domain of CD45.

CD45 contains tandem repeated protein tyrosine phosphatase (PTP) domains and is essential for the initiation of the earliest activation events resulting from Ag ligation of the TCR. The second PTP domain (D2) contains four CK2 phosphorylation sites in a unique 19-aa insert, which are targets of CK2 phosphorylation. This study was designed to evaluate the roles of these Ser residues in T cell activation. Transient transfection of the CD45- T cell line, J45.01, with CD45 cDNA incorporating four Ser to Ala (S/A) mutations in the 19-aa insert did not affect the magnitude of NF-AT activation resulting from TCR ligation. However, the basal level of NF-AT activity in unstimulated cells expressing the CD45 S/A mutation was elevated 9- to 10-fold. Increased basal NF-AT was dependent on extracellular Ca2+ stores as judged by EGTA treatment. In additional experiments, isolation of stable clones derived from transfection of the CD45 S/A mutant into CD45- H45.01 cells showed sustained calcium flux after TCR engagement. The sustained calcium flux returned to baseline levels after addition of EGTA, suggesting that the expression of the CD45 S/A mutant may have prevented deactivation of plasma membrane calcium channels. Consideration of both transient and stable transfection systems suggests that in addition to being essential for initial events in T cell triggering, the intact CD45 D2, 19-aa insert is necessary for regulation of TCR-mediated calcium signaling pathways.

Phosphorylation of CD45 by casein kinase 2. Modulation of activity and mutational analysis.

CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled the Vmax of CD45. CD45 isolated in vivo was highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible for in vivo phosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.

Identification of in vivo phosphorylation sites of CD45 protein-tyrosine phosphatase in 70Z/3.12 cells.

Phosphorylation of CD45, a transmembrane protein-tyrosine phosphatase (PTPase), has been proposed to mediate docking of signaling proteins and to modulate PTPase activity. To study the role of phosphorylation in CD45, in vivo phosphorylation sites of CD45 from 70Z/3.12 cells were identified using 32P labeling, trypsin digestion, two-dimensional peptide mapping, high performance liquid chromatography, phosphoamino acid analysis, matrix-assisted laser desorption/ionization mass spectrometry, and specific enzymatic degradation. Eight phosphopeptides, a through h, were isolated and four phosphorylation sites were identified. All four phosphorylation sites were in the membrane-distal PTPase domain (D2) and the C-terminal tail and none were in the membrane-proximal PTPase domain (D1). One site, Ser(P)939 peptide h, was in the D2 domain and, by comparison to the three-dimensional structure of PTP1B, is predicted to lie at the apex of the substrate binding loop. Ser939 was the only in vitro phosphorylation site for protein kinase C among the phosphorylation sites identified. Four of the C-terminal peptides identified (d, e, f, and g) spanned the same sequence and were derived from the same phosphorylation site in the C-terminal tail, Ser1204. Peptide a was derived from the intact C terminus and comprised a mixture of monophosphorylated peptides containing either Ser(P)1248 or Thr(P)1246. Knowledge of the precise phosphorylation sites of CD45 will lead to the design of experiments to define the role of phosphorylation in PTPase activity and in signaling.

LAR-PTPase cDNA transfection suppression of tumor growth of neu oncogene-transformed human breast carcinoma cells.

The incidence of amplification of neu oncogene-encoded protein tyrosine kinase in human breast cancer strongly supports the concept that protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in the proliferation, differentiation, and neoplastic transformation of breast epithelial cells. We examined the potential regulatory role of protein tyrosine phosphatases (PTPases) in the maintenance of cellular tyrosine phosphorylation by the introduction of leukocyte common-antigen-related PTPase (LAR-PTPase) cDNA into a tumorigenic human breast carcinoma cell line that overexpressed p185neu protein tyrosine kinase. The transfected human breast carcinoma cells expressed elevated levels of LAR-PTPase as assessed by reverse transcription-polymerase chain reaction and by analysis of LAR-PTPase protein. The LAR-PTPase-transfected human breast carcinoma cells had a significantly (P < 0.01) slower proliferation rate in vitro than control-transfected cells. When LAR-PTPase-transfected cells were inoculated into athymic nude mice, a consistent and significant (P < 0.05) suppression of tumor growth was observed. These results provide evidence that a specific PTPase, LAR-PTPase, can play a suppressive regulatory role in the tumor growth of human breast carcinoma cells that overexpress p185neu protein tyrosine kinase.

Elevation of lymphocyte CD45 protein tyrosine phosphatase activity during mitosis.

CD45 is an abundant cell-surface protein tyrosine phosphatase (PTPase) of hematopoietic cells that mediates specific tyrosine dephosphorylation in lymphocyte Ag activation. The hypothesis that CD45 PTPase activity varies during progression through the cell cycle was examined in this study. Expression of CD45 PTPase activity was determined in pre-B lymphoma (70Z/3.12) and cytolytic T lymphocyte (CTLL-2) cell lines after fractionation by counterflow centrifugal elutriation into cell cycle-stage-enriched subpopulations. For both cell lines, CD45 PTPase activity was elevated 2- to 10-fold in late G2 + M fractions compared with the PTPase activity of asynchronous cells. FACS and SDS-PAGE analyses indicated that there was only a minor increase in CD45 protein expression during progression through the cell cycle, suggesting that increased CD45 PTPase activity was a result of increased enzymatic activity. Cyclin B expression in 70Z/3.12 cells was evaluated in elutriated subpopulations and intact cyclin B was present in all cell cycle phases from G1 to late G2 + M, disappearing in mitosis and just before those fractions containing maximum CD45 activity. This observation indicated that the peak PTPase activity of CD45 occurred in late mitosis or in cytokinesis. The elevation of CD45 PTPase activity in mitosis supports the hypothesis that CD45 has a role in phosphorylation events occurring during cell division.

Increased expression of specific protein tyrosine phosphatases in human breast epithelial cells neoplastically transformed by the neu oncogene.

Protein tyrosine phosphorylation/dephosphorylation is a fundamental mechanism in the regulation of cell proliferation and neoplastic transformation; this metabolic process is modulated by the opposing activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPases). While the role of protein tyrosine kinases has been examined extensively in human breast tumorigenesis, the role of PTPases in this process is virtually unknown. To address this issue, an activated neu oncogene was introduced into an immortalized nontumorigenic human breast epithelial cell line (184B5). This resulted in a substantial increase in P185neu expression, which led to the formation of progressively growing carcinomas after such cells were inoculated into athymic nude mice. Importantly, a striking increase in the expression of specific PTPases, LAR and PTP1B, was observed in 3 independently neu transformed cell lines and their derived tumors. This elevation was verified at both the mRNA and protein levels. TC-PTP PTPase expression was only slightly increased in these neu transformed cells, and no expression of CD45 PTPase was observed. The level of neu expression, as well as the differential expression between P185neu and LAR/PTP1B, directly correlated with tumorigenicity. Furthermore, rat mammary carcinomas with elevated neu expression (neu-induced) also had sharply elevated LAR-PTPase expression when compared to rat mammary carcinomas with little or no neu expression (7,12-dimethylbenzanthracene induced); the level of expression of LAR PTPase was directly correlated with the level of neu expression. Thus, our results provide the first evidence that, in human breast carcinoma cells and in rat mammary carcinomas that have an induced increase in neu expression, a consistent and substantial increase in the expression of specific PTPases occurs. The relationship between P185neu-protein tyrosine kinase expression and specific PTPase expression may play a critical role in human breast tumorigenesis.

Lineage switch macrophages can present antigen.

Recent reports of "lineage switching" from a lymphoid to macrophage phenotype have left unresolved the question of whether such cells are functional macrophages or nonfunctional products of differentiation gone awry. This study demonstrates that several "macrophage-like" cell lines derived from v-Ha-ras-transformed pre-B cells have gained the capacity to effectively present antigen in MHC-restricted fashion. Using an assay involving the cocultivation of putative antigen-presenting cells with chicken ovalbumin (cOVA) and a cOVA-specific T-cell hybridoma, "lineage switch" cell lines were found to present antigen as effectively as macrophage-containing peritoneal exudates. Neither the original pre-B-cell precursors nor B-cell lymphomas derived from them present antigen. Thus, we have demonstrated that these "lineage switch" macrophages are capable of antigen presentation, a mature differentiated function. While gaining macrophage characteristics, these cells have also rearranged their kappa light-chain immunoglobulin locus, suggesting that macrophage differentiation and immunoglobulin rearrangement are not mutually exclusive processes. The existence of both lymphoid and myeloid characteristics in a cell fully capable of antigen presentation suggests greater plasticity in hematopoietic lineage commitment than conventionally thought to be the case.

Growth of MCF-7 human breast carcinoma in severe combined immunodeficient mice: growth suppression by recombinant interleukin-2 treatment and role of lymphokine-activated killer cells.

The severe combined immunodeficient (SCID) mouse, lacking functional T and B lymphocytes, has been considered by many groups to be a prime candidate for the reconstitution of a human immune system in a laboratory animal. In addition, this immuno-deficient animal would appear to have excellent potential as a host for transplanted human cancers, thus providing an exceptional opportunity for the study of interactions between the human immune system and human cancer in a laboratory animal. However, because this animal model is very recent, few studies have been reported documenting the capability of these mice to accept human cancers, and whether or not the residual immune cells in these mice (e.g. natural killer, NK, cells; macrophages) possess antitumor activities toward human cancers. Thus, the purpose of this study was (a) to determine whether or not a human breast carcinoma cell line (MCF-7) can be successfully transplanted to SCID mice, (b) to determine whether or not chronic treatment of SCID mice with a potent lymphokine (recombinant interleukin-2, rIL-2) could alter MCF-7 carcinoma growth, and (c) to assess whether or not rIL-2-activated NK cells (LAK cells) are important modulators of growth of MCF-7 cells in SCID mice. To fulfill these objectives, female SCID mice were implanted s.c. with MCF-7 cells (5 x 10(6) cells/mouse) at 6 weeks of age. Six weeks later, some of the mice were injected i.p. twice weekly with rIL-2 (1 x 10(4) U mouse-1 injection-1). Results clearly show that MCF-7 cells can grow progressively in SCID mice; 100% of the SCID mice implanted with MCF-7 cells developed palpable measurable tumors within 5-6 weeks after tumor cell inoculation. In addition, MCF-7 tumor growth was significantly (P less than 0.01) suppressed by rIL-2 treatment. rIL-2 treatment was non-toxic and no effect of treatment on body weight gains was observed. For non-tumor-bearing SCID mice, splenocytes treated in vitro with rIL-2 (lymphokine-activated killer, LAK, cells) or splenocytes derived from rIL-2-treated SCID mice (LAK cells) had significant (P less than 0.01) cytolytic activity toward MCF-7 carcinoma cells in vitro. In contrast, splenocytes (LAK cells) derived from tumor(MCF-7)-bearing rIL-2-treated SCID mice lacked cytolytic activities toward MCF-7 cells in vitro. No significant concentration of LAK cells in MCF-7 human breast carcinomas ws observed nor did rIL-2 treatment significantly alter growth of MCF-7 cells in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental expression of CD45 alternate exons in murine T cells. Evidence of additional alternate exon use.

The CD45 glycoprotein family exhibits cell line-age-associated structural heterogeneity arising in part from alternate 5' exon shuffling. Previous studies of exons involved in the final glycoprotein structure have provided evidence of alternate exon use for only three exons (Ex-4, 5 and 6). However, our prior data using reverse transcription-polymerase chain reaction (RT-PCR) suggested the presence of at least one additional CD45 alternate exon. By using RT-PCR, Southern blotting with exon-specific or exon splice junction-specific oligonucleotide probes and direct DNA sequencing of RT-PCR products, we demonstrated additional alternate use involving Ex-7. PCR analysis of stage I thymocytes (CD4-CD8-) revealed only faintly detectable bands for two isoforms: one lacking Ex-4, 5, 6 and 7 (a "minus-one" [Ex(-1)] isoform), and a smaller isoform preliminarily characterized as also lacking Ex-8. Stage II thymocytes (CD4+CD8+) prominently expressed both Ex(-1) and zero alternate exon (Ex(0] isoforms, with one exon (Ex(1] and two exon (Ex(2] isoforms also present. Among stage III thymocytes, both CD4+CD8- and CD4-CD8+ cells expressed only Ex(-1) and Ex(0) isoforms. CD45 alternate exon use in resting CD4+ and CD8+ lymph node T cells was divergent, with CD8+ cells additionally expressing an Ex(2) isoform. Among alloreactive T cell clones, band intensity for the Ex(1) isoform in CD4+ BC-3 cells was much less than for resting CD4+ T cells, while the CD8+ CTL clone 8.2.2 exhibited production of the higher alternate exon isoforms, Ex(2) and Ex(3). We conclude that at least four and possibly five alternate exons exist in the CD45 glycoprotein family, with a previously unrecognized isoform lacking Ex-4, 5, 6 and 7 prominently expressed in T cells. Shuffling of CD45 alternate exons appears to occur in an organized and predictable sequence during cellular maturation and activation.

Characterization of the CD45 molecule on murine intestinal intraepithelial lymphocytes.

The CD45 molecule was analyzed from murine intestinal intraepithelial lymphocytes (IEL). Immunofluorescent staining of CD8+ IEL revealed varying degrees of reactivity with mAb specific for CD45-restricted determinants, some which are typically expressed only by B cells. Immunoprecipitation of CD45 molecules from IEL yielded an array of proteins with apparent (m.w.) ranging from 180,000 to 260,000. The m.w. 260,000 form was restricted to IEL, was distinct from the B220 molecule, and was the only CD45 isoform that expressed the CD45-associated carbohydrate differentiation Ag CT1. Moreover, the CT1 determinant was present on cells of the Thy-1- but not the Thy-1+ IEL subset. Sequential immunoprecipitation studies indicated that expression of the m.w. 260,000 protein was not restricted to CT1+ cells. The protein composition of the m.w. 260,000 CD45 isoform was examined by using the polymerase chain reaction for analysis of CD45 variable exon usage. In contrast to B cells in which the major CD45 mRNA contained all three variable exons (exons 4, 5, and 6), IEL CD45 mRNA contained significant amounts of two-exon, single exon, and zero variable exon forms. Restriction enzyme analysis identified the single exon form as exon 5 and the two-exon form as a mixture of exons 4 and 5 and exons 5 and 6. Metabolic labeling of CD45 in pulse-chase experiments suggested that the generation of this high m.w. protein was caused by post-translational modifications, perhaps glycosylation. Overall, the results indicated that the high m.w. form of CD45 and the addition of the CT1 determinant were generated via IEL-specific post-translational modifications and not by novel alternate exon usage.

T200 alternate exon use in murine lymphoid cells determined by reverse transcription-polymerase chain reaction.

T200 glycoproteins of lymphoid and myeloid cells exhibit cell lineage-specific structural heterogeneity. Peptide heterogeneity appears to arise from alternate 5'-exon use (Ex-4, 5, and 6), potentially giving rise to eight distinct forms of T200 mRNA containing 0 to 3 of these alternate exons. A method is described for determining the number and identity of the three alternate T200 exons expressed in cells by using the polymerase chain reaction (PCR) and the reverse transcription-polymerase chain reaction (RT-PCR) without prior purification of RNA. Synthetic primers flanking the alternate exon region of T200 were designed to yield products for each possible exon combination having unique size and restriction enzyme sites. PCR amplification of plasmids containing T200 cDNA with none (pLy-5-68) or all three (p70Z/3-3) known alternate exons resulted in the amplification of 186 and 603 bp products, respectively. That amplified products were derived from T200 cDNA was verified by restriction enzyme mapping of each PCR product. T200 cDNA prepared from cell lines utilizing no alternate exons (BW5147) or all three exons (70Z/3.12) were analyzed by RT-PCR and contained amplified products of 186 bp (zero alternate exons) and 603 bp (containing Ex-4+5+6), respectively. RT-PCR of EL4 cells revealed approximately 186 and 330 bp products suggestive of zero and one alternate exon forms. Restriction mapping confirmed that EL4 cells contained a zero-exon form and a one-exon form containing Ex-5. Analysis of the 3B3 pre-B cell line yielded 186, 330, 460, and 603 bp products; restriction mapping revealed T200 mRNA for a zero alternate exon form, two distinct one- and two-exon forms (Ex-4; Ex-5; Ex-4+5; Ex-5+6), and a three-exon form (Ex-4+5+6). Other lymphoid cell lines were heterogeneous in T200 alternate exon use, with distinct patterns distinguishing B and T cells. RT-PCR can facilitate the analysis of variations in T200 alternate exon use among developmentally and functionally distinct lymphoid and myeloid cells.

Adherent lymphokine-activated natural killer cells in normal and severe combined immunodeficiency mice: large granular lymphocytes with natural killer cell phenotype and high cytolytic activity.

Plastic-adherent lymphokine-activated natural killer (LANK) cells were generated from nylon wool-nonadherent murine splenocytes cultured in recombinant interleukin-2 (IL-2). Under such conditions, adherent lymphokine-activated killer cells capable of killing natural killer (NK)-resistant targets were not generated. Adherent LANK cells proliferated rapidly and closely resembled NK cells in their morphology, cytotoxic reactivity, and surface marker expression. Mice with severe combined immunodeficiency (scid) were used to generate adherent LANK cells to define the role of T cells in LANK cell development. Scid lymphocytes responded to IL-2 by becoming adherent LANK cells with potent NK-like activity, suggesting that soluble lymphokines other than IL-2 that may have been produced by T cells were not required for the generation of LANK cell activity in mice.

Poly-N-acetyllactosamine structures on murine cell surface T200 glycoprotein participate in natural killer cell binding to YAC-1 targets.

Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.

Surface Ly-5 glycoprotein in murine natural killer (NK) cell development, target binding and cytotoxicity.

The role of surface Ly-5 glycoprotein expression in the binding and lysis of susceptible tumor targets by natural killer cells was studied using NK cell-enriched splenocytes from 6-8 week old C57BL/6 mice which were reacted with anti-Ly-5 serum in the presence or absence of a source of complement. A conjugate assay was used to demonstrate that abrogation of tumor cell lysis by anti-Ly-5 serum involved the inhibition of NK cell binding to susceptible YAC-1 targets. Additionally, reconstituted membrane vesicles from NK cell-enriched splenocyte populations blocked binding of effector cells to YAC-1 lymphoma targets, a phenomenon which was abrogated by pretreatment of vesicles with anti-Ly-5 serum. Indirect immunofluorescent labeling and cell sorting were used in the physical separation of Ly-5+ and Ly-5- cells to examine the effect of interferon and interleukin preparations on Ly-5 expression and Nk activity. Three hour treatment of sorted Ly-5- cells with murine alpha + beta interferon resulted in conversion of 22% of the cells to an Ly-5+ phenotype, as well as a significant increase in the percent specific lysis of NK-susceptible YAC-1 targets when compared to freshly sorted Ly-5- cells (29.5 +/- 1.9 vs 2.6 +/- 4.0; p less than .001). In vitro proliferation of sorted Ly-5- cells was induced by three week culture in an interferon- and interleukin-containing supernatant from ConA stimulated BALB/c splenocytes (CM), followed by repeat analysis of Ly-5 expression and cytotoxic activity. Cell sorter purified Ly-5- cells cultured in CM acquired substantial surface Ly-5 with concomitant high levels of cytotoxic activity that remained partially susceptible to inhibition by anti-Ly-5 serum. The data presented suggest that surface Ly-5 glycoprotein expression is important for binding of freshly isolated NK cells to YAC-1 targets. In addition, Ly-5- precursors of NK cells are present in murine splenic tissues and can be induced by CM to become highly active effector cells with increased surface Ly-5 expression. The persistent susceptibility of a subset of these cells to inhibition of cytotoxic activity by anti-Ly-5 serum provides additional evidence of an important role for the Ly-5 glycoprotein in the natural killer cell cytolytic mechanism against certain targets.

Poly-N-acetyllactosamine alterations of Thy-1 glycoprotein in lymphocyte differentiation.

Developmental changes in murine lymphocyte Thy-1 include both quantitative and qualitative alterations involving N-linked oligosaccharides. Comparison of immature with mature T-cells has shown that the oligosaccharides of Thy-1 are characterized by an increase in the number of sialic acid residues responsible for the acidic pI of peripheral T-cell Thy-1, and a decrease in those oligosaccharides responsible for Mr heterogeneity of thymocyte Thy-1. The research reported here suggests that the basis of the large Mr heterogeneity in Thy-1 of immature T-cells is the presence of repeating N-acetyllactosamine (R'Gal beta 1,4GlcNAc beta 1,3R") units in the oligosaccharide portion of the molecule. Lymphocytes were surface iodinated and 125I-thy-1 was purified by immunoprecipitation and preparative nonequilibrium pH gradient electrophoresis. The minimal Mr of unglycosylated Thy-1 after endoglycosidase F digestion was 15,000-16,000. Digestion of Thy-1 with endo-beta-galactosidase suggested that the complex type N-linked glycans in thymocytes, but not in lymph node T-cells, contained increased levels of polylactosamine. The presence of polylactosamine was confirmed by binding to a Datura stramonium lectin column which retarded and bound approx. 50% of thymocyte Thy-1 and only about 18% of lymph node T-cell Thy-1. Affinity chromatography using anti-i antibody immobilized on agarose beads indicated that the polylactosamine is probably present in a predominantly linear form. Since alterations of polylactosamine structures have been implicated in development and transformation in several systems, the present results suggest an important role for these glycans in immune-cell differentiation.

Interaction of gangliosides with B cells in splenic fragment cultures.

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.

Molecular weight and charge heterogeneity of Thy-1 glycoprotein in different populations of T-cells.

Molecular weight and charge microheterogeneity of Thy-1 glycoprotein expressed on different T-cell populations were compared by two-dimensional gel electrophoresis and endoglycosidase treatment. Thy-1 was immunoprecipitated from detergent lysates of radioiodinated T-cells of spleens, lymph nodes, Peyer's patches, peripheral blood, IL-2-cultured thymocytes and peanut agglutinin (PNA) separated thymocytes. In general, thymocytes and IL-2-cultured thymocytes expressed the highest levels of Thy-1 with a large Mr and charge variation. The Thy-1 of these cells was basic and contained low levels of sialic acid. On the other hand, peripheral T-lymphocytes exhibited a restricted Mr and more limited charge heterogeneity with higher amounts of sialic acid. The Thy-1 glycoprotein of mature, low Thy-1 PNA- thymocytes had Mr and charge heterogeneity identical to peripheral T-lymphocytes. The Mr and charge variation of immature PNA+ thymocytes was essentially identical to that of whole thymocytes. The molecular source of Mr heterogeneity in thymocyte Thy-1 and restricted Mr in lymph node Thy-1 was studied by analysis with endoglycosidase-H (Endo-H) and Endo-D. The results of Endo-H digestion showed that most of the Thy-1 polypeptides from both thymocyte and lymph node contained one high-mannose type oligosaccharide which was relatively small and not heterogeneous with respect to Mr. The complex-type oligosaccharides (Endo-D-sensitive) were larger and were responsible for the broad Mr-heterogeneity found in thymocyte Thy-1. Both thymocyte and lymph node Thy-1 generally appear to contain three N-linked oligosaccharides (one high-mannose and two complex) and sequential hydrolysis with Endo-H and Endo-D resulted in an unglycosylated polypeptide with an Mr of approx. 12,500.