Showing papers in "International Immunology in 1990"

Production of cytokines by mouse B cells: B lymphomas and normal B cells produce interleukin 10.

Abstract: We have examined a panel of murine Ly-l+ B lymphomas and purified normal murine peritoneal B cells separated into subsets on the basis of expression of the Ly-1 surface antigen, for their ability to produce cytokines. Where possible, we have used a combination of cytokine detection methods in order to compensate for differences In sensitivity and specificity, and the possibility of Inhibitors masking an activity. All the lymphomas tested were shown to constitutively express TGF-β and CSIFIIL-10. In addition, varying levels of IL-6, TNF-α and TNF-β, and G-CSF, were demonstrable In most of the lymphomas, and variants of one lymphoma (CH12) additionally produced varying levels of IL-α, IL-β, and GM-CSF. FACS purlfled normal Ly-1 + and Ly-lperltoneal B cells, were also shown to express RNA encdlng CSiFIIL-10, ILS, TNF-α and TNF-β, and very low levels of G-CSF, following stimulation with LPS. These data were supported by the detection of IL-6 and CSIFIIL-10 In supernatants from LPS-stimulated Ly-l+ and Ly-1− B cells using specific Immunoassays. None of the lymphomas or B cell preparations produced IL-la, IL-2, IL-5, IL-7, or IFN-γ. The purity of our normal B cell populations was assessed by phenotypic analysis on the FACS and also by the disappearance of certain mRNA transcripts after purification, e.g. CD4, c-fms, GM-CSF, and IFN-γ, most of which could be detected In LPSstimulated total peritoneal cell populatlons. This suggested that our B cell purification method had reduced, to a level undetectable In our assays, contaminatng T cells (CD4), macrophages (c-fms, GM-CSF), and NK cells (IFN-y). Absence of IL-3, IL-4, IL-5, and GM-CSF expression by LPS-stimulated Ly-l+ and Ly-1− B cells reduced the concern that contaminating peritoneal mast cells could account tor the observed cytoklne production. We therefore believe our data provide strong support for production of a subset of cytokines by LPS-stimulated normal B cells. Both the Ly-1+ B lymphomas and normal Ly-1+ and Ly-1+ B cells appear capable of expressing IL-6, TNF-α, TNF-β, and CSIFIIL-10. Thus, while other cytoklnes expressed only by the B lymphomas, e.g. IL-3, IL-4, and GM-CSF (although restricted to the CHI2 lymphomas) may be a resun of transformation, IL-6, TNFα, TNF-β, and CSIFIIL-10 may be important B cell derived immunoregulatory molecules In normal Immune responses Including B cell medlated antigen presentation, autocrine growth of B cells, or B cell medlated irnmunosuppression.

Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity.

Abstract: Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the context of I-Au. A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-alpha) mRNA and biological activity on L929 cells and their capacity to induce paralysis, the clinical hallmark of experimental allergic encephalomyelitis (EAE). No correlation was found between interleukin-2 or gamma interferon production and encephalitogenicity. LT and/or TNF-alpha may play a central role in the pathogenesis of EAE.

Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells.

Abstract: We have produced four mAbs reactive with the rat homolog of ICAM-1 (intercellular adhesion molecule-1), and presented evidence to indicate that the ICAM-1 molecule plays a differential role in the binding between high endothelial cells and lymphocytes, depending on the state of lymphocyte activation. The conclusion that the four antibodies (1A29, 6A22, 10A25, 10A56) specifically recognize the rat ICAM-1 homolog was based on: (i) their ability to inhibit homotypic aggregation of PHA-blasts; (ii) SDS-PAGE analysis of the antigen recognized; (iii) antigen distribution as assessed by immunoperoxidase staining of frozen sections; and (iv) cytokine-induced up-regulation of the antigen. Involvement of the ICAM-1 molecule in lymphocyte binding to high endothelial cells, the vital step in lymphocyte extravasation in lymph nodes, was examined by the use of one of the newly developed antibodies, and it was found that binding of activated but not resting lymphocytes to cultured high endothelial cells was significantly blocked by the anti-ICAM-1, implying that ICAM-1 has differential involvement in the adherence of lymphocytes to high endothelial cells and that it may play an important role in dissemination of memory lymphocytes to systemic circulation by facilitating or strengthening the binding to the specialized postcapillary high endothelial venules (HEV).

HLA-A- and HLA-B-specific monoclonal antibodies reactive with free heavy chains in Western blots, in formalin-fixed, paraffin-embedded tissue sections and in cryo-immuno-electron microscopy

Abstract: The mAb HCA2 reacts preferentially with HLA-A locus heavy chains. Its reactivity contrasts with that of HC10, a mAb with more pronounced specificity for HLA-B and -C heavy chains. Both HCA2 and HC10 were raised against free class I heavy chains of HLA-A and -B antigens respectively, to obtain mAbs that would still react with denatured class I antigens, as they occur in Western blotting, conventional light microscopical analysis of formalin-fixed, paraffin-embedded sections, and cryo-immuno-electron microscopy. HCA2 and HC10 indeed retain strong reactivity with free class I heavy chains in Western blots. The HCA2 mAb in particular reacts in a locus-specific manner by biochemical criteria. Conditions are described for use of HCA2 and HC10 in immunohistochemical staining of formalin-fixed, paraffin-embedded sections. HCA2 and HC10 also produce strong reactivity in immuno-electron microscopy. Their use allows the determination of tissue and subcellular distribution of class I antigens.

Distribution of the CD68 macrophage/myeloid associated antigen

Abstract: The distribution of the pan-macrophage CD68 antigen, recognized by six different monoclonal antibodies, was examined in human blood, tissue, and cell lines using APAAP staining and Western blotting. All antibodies stained monocytes and macrophages, but labelling of neutrophils, basophils, and lymphocytes was seen with some of the reagents. In addition, the CD68 antibodies demonstrated a variety of staining patterns on some non-haemopoietic cells. The subtle differences between the reactions of the different antibodies suggested that the CD68 antigen may be heterogeneous, possibly due to differences in glycosylation. While CD68 antibodies are very useful markers of the macrophage/myeloid series, the presence of small amounts of the antigen on some lymphoid and non-haemopoietic cells means that care should be taken when using them for the diagnosis of tumours of unknown origin.

All T15 Id-positive antibodies (but not the majority of VHT15+ antibodies) are produced by peritoneal CD5+ B lymphocytes.

Abstract: After adult irradiation and reconstitution with autologous bone marrow, BALB/c and C.B20 mice no longer utilize the T15 Id in response to phosphorylcholine. T15 Id expression can be restored by transfers of peritoneal B cells or by FACS-purified CD5+ IgM+ lymphocytes (but not by T lymphocytes) from syngeneic donors. Using bone marrow and peritoneal cell donors that are congeneic for heavy and light chain allotypes, the exclusive origin of the T15 Id in peritoneal B cells was ascertained. These conclusions have been essentially confirmed by immunization with either anti-T15 Id or anti-VHT15 antibodies conjugated to lipopolysaccharide. Thus, the production of VHT15-positive antibodies continues at control levels in bone marrow-reconstituted animals while no T15 Id production can be stimulated even in this protocol of direct B cell stimulation. These results constitute the first formal demonstration of the exclusive production of and Id by CD5+ B-cells.

Structure and evolution of mammalian VH families.

Abstract: Antibodies are encoded by a limited number of germline gene segments that undergo somatic diversification through rearrangement and mutation. Because these mutation processes are efficient, it is widely believed that there is little environmental selection pressure for the maintenance of specific antibody gene sequences. We have performed pairwise comparisons of known germline (as opposed to somatically generated) antibody VH elements with the hope of identifying conserved structural features common to sets of VH gene segments. These studies reveal that VH families arose prior to the mammalian radiation and have since been conserved, that this conservation appears to reflect selection at the level of protein sequence, and that the conserved regions are discretely localized on a solvent-exposed face of the heavy chain, at some distance from the antibody combining site. A family-specific region was also identified within the recombinase recognition sequences. Our results provide a context for theories that address the physiological significance of variations in VH family utilization during the development of the immune repertoire.

Are primary alloresponses truly primary

Abstract: Proliferative T cell responses against major histocompatibility complex (MHC) incompatible stimulator cells in the mixed lymphocyte reaction are conventionally regarded as primary. However, it is generally accepted that the recognition of allogeneic MHC products results from a cross-reaction by self-MHC-restricted cells. These two assumptions were tested by examining the contribution of previously primed and naive T cells to 'primary' alloresponses. Peripheral blood T cells were separated into LFA-3+, memory, and LFA-3-, naive, populations by fluorescence-activated cell sorting. In contrast, to recall antigen responses to Candida albicans which were almost entirely confined to the LFA-3+, memory, population, the proliferative response to MHC incompatible stimulator cells, including HLA-DR-expressing mouse L cell transfectants, was equally distributed between the two T cell subsets in 5 day assays. Furthermore, limiting dilution analysis showed that the frequency of alloreactive T cells did not differ significantly between the two populations. The kinetics of proliferation in the two populations differed but were consistent with their naive and memory phenotype, in that after 3 days of culture the LFA-3+ cells proliferated more strongly to MHC alloantigens. These results show that a substantial proportion of 'primary' alloresponses are contributed by previously primed cells. In addition, the evidence for the cross-reactive hypothesis is supported and extended from the clonal to the population level.

Degenerate binding of immunogenic peptides to HLA-DR proteins on B cell surfaces

Abstract: Binding of linear fragments of protein antigens to class I or class II molecules of the MHC is necessary for the stimulation of a cellular immune response. This report describes the binding of a biotinylated T cell determinant from influenza hemagglutinin to class II proteins on the surface of Epstein-Barr virus-transformed B lymphocytes. The rapid, simple, and quantitative binding assay involves flow cytometric analysis of transformed B cells stained with fluoresceinated streptavidin following incubation with the biotinylated peptide. Binding of the biotinylated peptide required cell surface expression of human class II molecules, and was inhibited by an anti-HLA-DR monoclonal antibody as well as the unbiotinylated natural determinant. Rates of association and dissociation of the peptide were similar to those reported for purified MHC class II proteins, and the peptide bound only approximately 1% of the DR molecules expressed on the cell surface. When assayed on many different DR-homozygous B cell lines, the biotinylated hemagglutinin T cell determinant bound to HLA-DR on each cell line. The degeneracy of peptide binding to B cell lines was not unique to the hemagglutinin peptide because three other biotinylated T cell determinants failed to bind to class II deficient B-lymphoblastoid cells but bound to varying degrees to multiple DR-homozygous lines.

Molecular cloning of cDNA encoding MS2 antigen, a novel cell surface antigen strongly expressed in murine monocytic lineage

Abstract: cDNA clones which strongly hybridized with a 3.1 kb mRNA from mouse macrophages and macrophage cell lines and weakly with mRNA from P815 but not from a variety of other cell lines and tissues were isolated from cDNA libraries constructed using mRNA from murine macrophage cell lines and peritoneal macrophages. Treatment of a macrophage cell line with macrophage stimulators significantly enhanced transcription of the mRNA. Sequencing analysis of these clones demonstrated that the cDNA consisted of 3036 bp insert containing a 2478 bp open reading frame followed by a 538 bp 3' untranslated region. The amino acid sequence, deduced from the nucleotide sequence of the cDNA, predicted a protein containing a signal peptide, an extracellular region, a transmembrane domain, and a cytoplasmic tail. The extracellular region had five putative N-glycosylation sites and a cysteine-rich domain, whereas the cytoplasmic region consisted of a proline-rich amino acid sequence significantly similar to CD2. SDS-PAGE and NEPHGE SDS-PAGE analysis of the immunoprecipitated membrane of the macrophage cell lines prepared by using rabbit anti-MS2 peptide antibody raised against a synthetic peptide preparation relative to a hydrophilic region of the MS2 amino acid sequence confirmed that MS2 protein is a cross-linked protein having approximate molecular sizes of 89 kd and pl 6.5-7.0. These results show that MS2 protein is a novel cell surface antigen expressed mainly in monocytic lineages.

Molecular cloning of a cDNA encoding the human interleukin 4 receptor

Abstract: Using the mouse interleukin 4 (IL-4) receptor cDNA as a probe, we isolated a cDNA encoding the human IL-4 receptor (hIL-4 receptor) from a multifactor-responsive human myeloid cell line, TF1. The cDNA encodes for an open reading frame of 825 amino acids including a signal sequence (25 amino acids), the external domain (207 amino acids), a transmembrane domain (24 amino acids), and a large cytoplasmic domain (569 amino acids). The human IL-4 receptor has a 65% identity with the mouse IL-4 receptor at the nucleic acid level and retains the typical structural motif of the previously described cytokine receptor family. COS7 cells transfected with the full-length cDNA expressed high levels (140,000 sites/cell) of IL-4 binding sites, with a Kd = 80 pM, an affinity identical to that of the original TF1 cells. Similar to IL-4 responsive cells, cross-linking of [125I]IL-4 to COS7 cells transfected with the cDNA showed a major protein of 130-150 kd and minor species of 55-85 kd.

High frequency of memory and effector gag specific cytotoxic T lymphocytes in HIV seropositive individuals.

Abstract: Human immunodeficiency virus type 1 (HIV-1) specific cytotoxic T lymphocytes (CTL) have been detected in most patients following infection. It has also been suggested that the specific CTL response, which may play an important role in delaying disease in infected humans, may decline during the course of disease progression. We have measured HIV peptide specific CTL precursor frequency by limiting dilution analysis in two healthy seropositive patients whose fresh peripheral blood mononuclear cells (PBM) specifically lysed MHC matched target cells (restricted by HLA B27 and B8 respectively). The frequency of HIV peptide specific CTL precursors is high (3 x 10(-3)-10(-4], but lower than estimates of circulating CTL with the same specific cytotoxic activity present directly in peripheral blood using the same sample (10(-2)-10(-3]. We suggest that this difference may result from terminal effector CTL differentiation in the T cell lineage. This implies that only a subset of CTL effectors have growth potential, whereas relatively higher levels of CTL with lytic activity can be detected in PBM of healthy HIV seropositive patients.

Structural homologies between two HLA B27-restricted peptides suggest residues important for interaction with HLA B27.

Abstract: Recently we described an HLA B27-restricted peptide derived from HIV gag p24 protein. In this study we have isolated an HLA B27-restricted peptide from the nucleoprotein (NP) of influenza A virus. The shortest fragment recognized by cytotoxic T lymphocyte (CTL) is eight amino acids long, residues 384-391. Comparison of the sequence of these two HLA B27 restricted peptides reveals homologies which can be aligned from one peptide to the other. Of the eight residues, two are identical: tryptophan and isoleucine. Both peptides have a positively charged residue at the N terminus, lysine at position 265 of gag and arginine at position 384 of NP. Using modified peptides we have shown that lysine or arginine is crucial for the interaction with HLA B27. The wild-type gag peptide blocked CTL recognition of NP peptide by influenza-specific CTL, but removal of the lysine prevented inhibition of NP peptide recognition. The importance of these charged residues was confirmed by the observation that truncated NP and gag peptides where the lysine or arginine was removed were not recognized by specific CTL. Further studies showed that the tryptophan residue influenced the association of the gag peptide with HLA B27, because the affinity of the gag peptide for B27 was strongly increased after replacing this residue with a leucine or a tyrosine. However, these peptides were not recognized by gag-specific CTL, suggesting that the tryptophan may interact with both HLA B27 and T cell receptor. These observations should help in the identification of HLA B27-restricted peptides from other viruses or organisms.

Persistence of memory B cells in mice deprived of T cell help.

Abstract: The influence of T cell help on the induction, maintenance, and recall of B cell memory to a T cell-dependent antigen was studied in mice. The antigen was a hapten coupled to a protein carrier, and helper T cells were eliminated before, during, or after immunization by treatment of the animals with anti-CD4 antibodies. B cell memory was quantified in an adoptive cell transfer system, transferring B cells from the primed animals together with carrier-primed syngeneic T cells. Anti-CD4 treatment completely inhibited the induction of primary and secondary responses and of B cell memory. In contrast, it did not affect established B cell memory over a period of at least 6 weeks. Consequently, this suggests that within this time range B cell memory to a T cell-dependent antigen does not rely on the continuous recruitment of naive B cells into the memory compartment, and memory B cells need little or no (antigen-mediated) T cell help in order to persist.

Expression of glycolipid receptors to Shiga-like toxin on human B lymphocytes: a mechanism for the failure of long-lived antibody response to dysenteric disease.

Abstract: Fresh and transformed human B lineage cells were found to be sensitive to the cytotoxic action of Shiga-like toxin (SLT), a bacterial cytotoxin. The toxin was specifically bound by the glycolipids globotriosylceramide and galabiosylceramide expressed on the surface of sensitive cells. Mutant Daudi cells selected for resistance to SLT cytotoxicity (SLTR20) were deficient in SLT-binding glycolipids and failed to bind SLT to their surface, suggesting a role for these glycolipids in the mediation of SLT cytotoxicity. Of a number of normal and transformed lymphoid and myeloid cells screened for SLT sensitivity, only B lymphoid cells were susceptible to SLT action. Moreover, B lymphoid cells were the only cells expressing the SLT binding glycolipids. In vitro B cell activation studies with Epstein-Barr virus and pokeweed mitogen both indicated that the vast majority of SLT-sensitive B cells belong to the IgG and IgA committed subset, whereas most IgM and IgM/D producing cells were resistant to SLT toxicity. The selective elimination of IgG and IgA committed cells may explain the production of only IgM class anti-SLT antibodies in Shigella-infected humans leading to the failure of long-term immunity to dysenteric disease.

Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause autoimmune hemolytic anemia by two distinct pathogenic mechanisms.

Abstract: In vivo pathological manifestations of eight monoclonal anti-mouse red blood cell (MRBC) autoantibodies obtained from unmanipulated NZB mice were determined in BALB/c mice. Three (two IgG1 and one IgG2a) of four IgG monoclonal antibodies (mAb) and two of four IgM mAb were able to induce anemia following their i.p. injection. All five pathogenic anti-MRBC mAbs reacted only with MRBC, whereas non-pathogenic anti-MRBC mAbs showed binding to different species of RBC. Competition studies suggested the presence of at least two distinct epitopes recognized by our pathogenic anti-MRBC mAb. Histological examinations revealed that anemia resulted from either marked sequestration of agglutinated MRBC in spleens and livers or erythrophagocytosis, most remarkably by Kupffer cells in livers. This difference was correlated with the ability of each mAb to mediate Fc receptor-dependent phagocytosis by macrophages. The absence of complement-mediated hemolysis in vitro and the development of anemia in C5-deficient or C3-depleted mice indicated a minor role, if any, for complement-mediated lysis in the anemia induced by our anti-MRBC mAb. Our results suggest that (i) at least two different pathogenic epitopes are implicated in autoimmune hemolytic anemia; and (ii) sequestration of agglutinated MRBC in spleens and livers and Fc receptor-dependent phagocytosis, but not complement-mediated hemolysis, are the major mechanisms for the development of autoimmune hemolytic anemia.

CD4+ CD8+ murine intestinal intraepithelial Iymphocytes

Abstract: We have studied a population of CD4+ intestinal intraepithelial lymphocytes using two-color flow cytometric analyses, and in highly purified fluorescent-activated cell-sorted preparations. Although CD4+ T cells present within the epithelial immune compartment comprised only approximately 10-20% of the total intestinal epithelial lymphoid cells, an unusually high proportion of those CD4+ lymphocytes expressed a CD4+CD8+ phenotype which is rarely encountered in peripheral T cells. By comparison, CD4+ lymphocytes from spleen or lymph nodes existed exclusively as single-positive T cells. Analyses of CD4 and CD8 expression on lymphocytes from Peyer's patches, the lamina propria, and IEL indicated that CD4+CD8+ lymphocytes were unique to the IEL. Using CD4+CD8+ preparations obtained by fluorescent-activated cell sorting, CD4+CD8+ epithelial T cells were found also to express CD3 and Thy-1 surface markers. This heretofore undescribed extrathymic population of double-positive T cells constitutes a unique peripheral T cell subset which may be involved in intestinal T cell maturation and development, or could represent a highly specialized effector population.

Structure and expression of germline immunoglobulin γ3 heavy chain gene transcripts: implications for mitogen and lymphokine directed class-switching

Abstract: We have characterized the structure and expression of transcripts synthesized from the murine germline immunoglobulin gamma 3 heavy chain gene in certain B-lineage cells. The transcripts initiate upstream of the switch gamma 3 region, generating a 5' exon that is spliced to C gamma 3 exons. Expression of this germline transcript is induced when splenic B cells or A-MuLV-transformed pre-B cell lines are cultured in the presence of lipopolysaccharide (LPS). Addition of interleukin-4 (IL-4) to these lipopolysaccharide (LPS) cultures dramatically inhibits induction of the germline gamma 3 transcript. Induction of germline gamma 3 transcripts occurs before the increased accumulation of gamma 3-producing cells and VDJ-gamma 3 mRNA in cultures of splenic B cells. These data provide further evidence that germline CH transcriptional units are important components in the regulation of heavy chain class-switching. In addition, the pre-B cell lines that we describe represent the first example of permanent cell lines that regulate expression of the germline gamma locus in response to LPS plus IL-4 treatment in a manner analogous to normal B cells; therefore these lines should represent an excellent model system to further study the molecular mechanisms by which germline expression is regulated by these agents.

Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells

Abstract: Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.

Ligand thresholds at different stages of T cell development.

Abstract: Certain T cell ligands can stimulate most or all T cells whose receptor is encoded by particular V beta genes. Exposure to these same ligands during intrathymic development leads to deletion of T cells bearing these same receptors. We have utilized one such ligand, staphylococcal enterotoxin B (SEB), to examine the quantitative differences in these two responses. By comparing the dose of SEB required to delete developing T cells in thymic organ culture with that required to stimulate spleen cells to expand clonally, we observe that clonal deletion is one to two orders of magnitude more sensitive to SEB. Because the T cell ligand involves not only SEB but also class II MHC molecules, and because the culture conditions are distinct, this system does not allow one to state that intrathymic T cells are intrinsically more sensitive to negative selection than peripheral T cells are to activation. However, these studies for the first time do establish a quantitative comparison of these two processes and suggest that there is a margin for error built into the clonal deletion process, such that ligands presented in the thymus are 30- to 100-fold more active in clonal deletion than is the same ligand in activation of peripheral T cells. This result agrees well with the observation that naturally occurring unknown ligands associated with I-E can clonally delete cells that are not activated by the same ligand in mixed lymphocyte culture.

Analysis of recent thymic emigrants with subset- and maturity-related markers.

Abstract: The thymus plays an integral role in the development and production of T lymphocytes However, thymocytes differ markedly in their phenotypic characteristics from the T cells normally found in the peripheral lymphoid organs We have examined the phenotypic characteristics of recent thymic emigrants and compared them with both mature phenotype thymocytes (CD4+ CD8-CD3+ and CD4-CD8+ CD3+) and lymph node T cells Recent thymic emigrants were defined as those fluorescein-positive cells found in the lymph node up to 16 h after intrathymic injection of fluorescein Most cells emigrating from the thymus expressed CD3 and either CD4 or CD8, indicating maturity Recent thymic emigrants, like mature phenotype thymocytes, were slightly larger on average than peripheral T cells, but this differential was lost within 24 h of emigration Also like mature thymocytes but unlike peripheral T cells, some recent emigrants expressed heat-stable antigen This did not change within 24 h of emigration The antigen CD44 (Pgp-1, Ly-24) was expressed on a proportion of mature thymocytes, recent thymic emigrants, and peripheral T cells, and its expression did not show any clear relationship to maturity The antigen CD45R also did not show marked changes associated with maturity, but our data do not parallel the published data of the expression of CD45R in the human We conclude that recent thymic emigrants are phenotypically mature with respect to some antigens but not others None of the antigens we investigated could have been used to uniquely distinguish recent thymic emigrants from peripheral T cells or from mature thymocytes

Disruption of thymocyte development and lymphomagenesis induced by SV4O T-antigen

Abstract: The lck gene encodes a membrane-associated protein tyrosine kinase that is expressed specifically in lymphoid cells, especially thymocytes. Structural analysis of the murine and human lck genes previously identified conserved 5' flanking sequences that were proposed to represent transcriptional regulatory elements. Here we demonstrate that a murine lck promoter construct containing these sequences directs the expression of the SV40 T-antigen gene in lymphoid cells. Remarkably, expression of SV40 T-antigen in transgenic animals dramatically disturbs thymic development, resulting in preferential loss of CD4+CD8+ thymocytes. In contrast, immature cells lacking both CD4 and CD8 markers are present in near-normal numbers. Thus SV40 T-antigen expression appears partially to arrest thymopoiesis. Mice bearing the lck-SV40 transgene develop readily explantable thymic tumors at 12-18 weeks of age. Fluorocytometric analyses of lck-SV40 tumor cells reveal that immature thymocytes are frequently immortalized. The lck-SV40 mouse may therefore provide materials for the in vitro investigation of thymocyte differentiation.

Kinetic study of a galactosyltransferase in the B cells of patients with rheumatoid arthritis.

Abstract: The sugar chains of IgG samples purified from sera of patients with rheumatoid arthritis (RA) contain many fewer galactose residues than those from sera of healthy individuals. Enzymatic studies revealed that the low galactose content in the IgGs of RA patients results from the reduced activity in the B cells of a galactosyltransferase (EC 2.4.1.90), which preferentially transfers galactose to asialo-agalacto-IgG. Asialo-agalacto-transferrin and asialo-ovine submaxillary mucin were also galactosylated by detergent-activated human B cell homogenates. However, no difference in the enzymatic activities toward these two acceptors was detected between the B cells from RA patients and from non-RA patients and healthy individuals. Enzyme kinetic studies revealed that an affinity of the galactosyltransferase in the B cells from RA patients was lowered for UDP-Gal but not for asialo-agalacto-IgG, while the affinities for UDP-Gal and asialo-agalacto-transferrin of the galactosyltransferase were not changed between the B cells from RA patients and from non-RA patients and healthy individuals in accordance with their enzyme activities. The results indicated that the reduced galactosyltransferase activity toward asialo-agalacto-IgG in the B cells from RA patients can be ascribed to the lowered affinity for UDP-Gal.

Somatic mutations in antibodies expressed by germinal centre B cells early after primary immunization

Abstract: We have studied the maturation of the immune response by looking at the generation of antibody diversity in germinal centre B cells. Mice were immunized with the antigen 2-phenyloxazolone. Germinal centre B cells, defined by their strong binding to peanut agglutinin (PNA(hi], were sorted from the spleen and fused. Ten days after immunization high numbers of antigen specific hybridoma lines were obtained from the PNA(hi) subset of B cells, suggesting that the small fraction of B cells which are PNA(hi) harbour the antigen activated population. The majority of the day 10 sequences from PNA(hi) cells were shown to be mutated. However, in contrast to results from later stages of the immune response, most of the mutations found were silent. The preferential expansion of B cell clones expressing the mutations characteristic of the mature response was not observed at this stage. Among these hybridoma lines was at least one which, apparently through somatic mutation, had lost the ability to bind antigen. We conclude that in the micro-environment of the germinal centre the B cell repertoire is diversified by hypermutation.

Cloning and sequence analysis of a novel beta 2-related integrin transcript from T lymphocytes: homology of integrin cysteine-rich repeats to domain III of laminin B chains.

Abstract: The integrins have been grouped into three subfamilies based on the utilization of three distinct beta chain subunits, beta 1, beta 2, and beta 3. The recent discovery of beta 4 and beta 5 subunits, and the sharing of alpha subunits between beta subfamilies reflects a greater structural and functional diversity of the integrin supergene family than first anticipated. We exploit integrin gene sequence homology for the identification of a new integrin beta chain. Oligonucleotide probes designed from the highly conserved Arg-Gly-Asp (RGD)-binding domain were used to amplify related transcripts from phytohaemagglutinin-activated human peripheral blood lymphocytes using the polymerase chain reaction (PCR). Five PCR products encoding beta 1, beta 2, beta 3, beta 4, and a new beta clone, designated beta 7, were identified. Full-length beta 7 cDNA was isolated from an activated human T lymphocyte library, using an oligonucleotide probe constructed from the beta 7 PCR product. The beta 7 cDNA contains a long open reading frame of 2391 bp which encodes a protein sequence consisting of 797 amino acids. The encoded sequence revealed a typical signal peptide, a predominantly hydrophilic 707 amino acid residue domain with 8 N-glycosylation sites, a transmembrane domain, and a C-terminal domain of 52 amino acids. The beta 7 sequence showed homology to known beta 1, beta 2, beta 3, beta 4, and beta 5 sequences of 43, 46, 38, 32, and 37% respectively. Four cysteine-rich homologous repeat sequences were found in beta 7 and were homologous to sequences in other integrin beta subunits, and to domain III of the laminin B chains. Part of this cysteine-rich region is homologous to proteins that contain epidermal growth factor-like repeat sequences. Northern analysis revealed that mature beta 7 mRNA is approximately 3.5 kb in size, and expression was restricted to T and B lymphocytes in the small panel of cell types examined. We conclude that beta 7 may be a new member of the leukocyte cell adhesion molecule subset of integrins, and is a candidate immunoregulatory molecule.

Genetic basis of the neonatal antibody repertoire: germline V-gene expression and limited N-region diversity.

Abstract: We report here on the molecular characterization of heavy and light chain V-regions of antibodies isolated from the highly connected idiotypic network of the newborn BALB/c mouse. Nucleotide sequence analysis of eight hybridomas confirmed their germline origin. Furthermore, in contrast to most hybridomas and myelomas derived from adult mice, the majority of these clones were found to lack N-region sequences. These data show that somatic processes amplifying junctional diversity are relatively inactive early in ontogeny, and that germline gene expression alone ensures idiotypic complementarities in the developing immune system.

Selection of VH gene repertoires: differentiating B cells of adult bone marrow mimic fetal development.

Abstract: In the present study we have compared by in situ hybridization and by a CFU-B colony assay VH family usage in the pre-B and B cell compartments of the bone marrow of adult BALB/c and C57BL/6 mice. We have found that the position dependent increased expression of the VH 7183 family, observed in neonatal mice, is characteristic of early differentiating B cells of adult mice. The quantitative analysis for each VH family of the number of pre-B and B cells produced daily and the number of mature B cells present in the peripheral immunocompetent cell pool of adult BALB/c demonstrates the existence of selecting mechanisms operating within the bone marrow at the level of the emergent repertoire and at the level of export of newly formed cells into the periphery. This selective process results in the decreased peripheral representation of the VH 7183 family and in the accumulation of cells belonging to the other VH families. Selection of VH family usage in peripheral repertoires may be determined according to lymphocyte life-span, as we have found a preferential utilization of the VH J558 family in populations of partially enriched, long-lived B cells.

The kinetics of immature murine thymocyte development in vivo.

Abstract: The dynamics of cell generation and turnover in the young adult murine thymus has been studied by in vivo administration of [6-3H]deoxythymidine, isolation of thymocyte subpopulations by negative depletion and cell sorting procedures, and assessment of dividing cells and their products by autoradiography. The flow of label through subpopulations of CD4-CD8- thymocytes defined by the markers heat stable antigen (HSA), phagocyte glycoprotein 1 (Pgp-1), interleukin 2 receptor (p55) (IL-2R), and CD3 was determined, to check the developmental sequence deduced from intrathymic transfer and molecular approaches. In addition, the flow of label 'downstream' into the CD4+CD8+ cortical populations was followed to check if cells expressing CD8 alone were obligatory intermediates. The main findings were: (i) support for the following sequence within the CD4-CD8- group: HSA++Pgp-1+IL-2R(-)----HSA++Pgp-1-IL-2R(+)----HSA++Pgp-1-IL- 2R-; (ii) the majority of cell generation and cell turnover within the CD4-CD8- population was due to the HSA++IL-2R-Pgp-1- subpopulation; (iii) the rate of cell output from the proposed intermediate CD3-CD4-CD8+ subpopulation was equivalent to only 55% of the cell output from its proposed precursor, the most mature CD4-CD8- subpopulation, suggesting that many double negatives differentiate directly (or via CD3-CD4+CD8- intermediates) into double positives; and (iv) the CD4-CD8-HSA- (and CD3+) thymic subpopulation contained very few cycling cells and turned over extremely slowly, indicating that these slowly accumulating product cells are off the mainstream of T cell development.

Characterization of murine complement receptor type 2 and its immunological cross-reactivity with type 1 receptor

Abstract: We have previously demonstrated that some mAbs prepared against mouse complement receptor type 1 (CR1) bind a 150,000 Mr protein in addition to the 190,000 Mr CR1 protein. We now identify the 150,000 Mr murine protein as complement receptor type 2 (CR2), since: (i) one of the monoclonal antibodies that bind this protein inhibits rosette formation between mouse B cells and C3d-bearing sheep erythrocytes; (ii) as is known for human CR2, this protein is present on B lymphocytes but not T lymphocytes; and (iii) this protein must have affinity for C3b, since it has weak factor I cofactor activity. In addition, this protein resembles the 145,000 Mr human CR2 molecule in size. Since four of the five mAbs that were produced by immunization with CR1 also bound CR2, and they bind to different CR1 epitopes, it seems that murine CR1 and CR2 share multiple epitopes. Injection of mice with one of the CR1-CR2 cross-reactive mAbs almost eliminated both CR1 and CR2 expression, but did not decrease B cell numbers or the expression of B cell IgM, Ia, or B220 antigens. In contrast, injection of mice with a non-cross-reactive anti-CR1 antibody only modulated CR1 expression. These antibodies should thus provide useful tools for the study of the in vivo roles of B cell complement receptors.

An associated molecule, p64, with high-affinity interleukin 2 receptor.

Abstract: TU11 mAb specific for the human interleukin-2 receptor (IL-2R) beta-chain, p75, co-precipitated two molecules, p64 and p55, with the beta-chain in the lysates of cells bearing the high-affinity IL-2R. The co-precipitation was detected in the presence of IL-2 even in the absence of a chemical cross-linker. H-48 mAb specific for the IL-2R alpha-chain completely pre-absorbed p64 as well as p55 and partially pre-absorbed the beta-chain from the lysates. The co-precipitation was also detected in phytohemagglutinin-stimulated normal peripheral blood lymphocytes, which express the high-affinity IL-2R, but not in the cell line cells bearing only the low-affinity IL-2R. The peptide maps indicate that p64 is a molecule distinct from both the alpha- and beta-chains, and that p55 is identical to the alpha-chain. These findings suggest that p64, along with the alpha- and beta-chains, is a component of the high affinity IL-2R complex, and we have tentatively named it the gamma-chain of IL-2R.