Search

Molecular studies of murine Fc gamma R have revealed much exciting new information about the structure and regulation of Fc gamma RI and Fc gamma RII genes and of the Fc gamma RI protein. The Fc gamma RI gene is composed of six exons, whereas the Fc gamma RII gene is composed of ten. The extracellular domains are encoded by individual exons in both genes (three in Fc gamma RI and two in Fc gamma RII); however, the Fc gamma RII gene shows greatest complexity in the region encoding the cytoplasmic tail and membrane spanning region, which is encoded by four exons compared to only one in the Fc gamma RI gene. Expression of Fc gamma RII is controlled by elements within the first 641 bases upstream of the transcription initiation site. The function of the domains of Fc gamma RI has been defined with the surprising finding that in the absence of the third domain the first two extracellular domains function as a broadly specific low affinity Fc gamma RII-like receptor.

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.

This study describes the production of soluble Fc gamma RII by a cell line, D1B1, obtained by transfection of mouse L cells with a murine beta 1 Fc gamma RII cDNA. Upon incubation at 37 degrees C, radioiodinated D1B1 cells release a 39-kDa soluble Fc gamma RII, reacting with the rat anti-mouse Fc gamma RII monoclonal antibody 2.4G2, and binding to mouse IgG2a, IgG2b and IgG1 but not IgG3. In contrast to the transmembrane 50- to 70-kDa receptor, this soluble Fc gamma RII does not react with antibodies directed against a peptide corresponding to the 15 carboxy-terminal intracytoplasmic amino acids of beta Fc gamma RIII. N-Glycosidase F treatment generates a 18-kDa polypeptide. A 32- to 40-kDa soluble Fc gamma RII, which resolves into 18.5- and 20-kDa polypeptides after deglycosylation, was also isolated from the culture medium of unlabeled D1B1 cells. Therefore, this study indicates that soluble Fc gamma RII corresponding to the two extracellular domains of Fc gamma RII are generated by cleavage of membrane Fc gamma RII. Proteolysis occurs most probably at the vicinity of the transmembrane region of the receptor, around amino acids 165 to 180.

The characteristic finding of high levels of circulating immune complexes in patients with the autoimmune connective tissue diseases systemic lupus erythematosus (SLE) or scleroderma has raised the possibility that these patients may have a primary defect in immune complex clearance. The Fc receptor for IgG (Fc gamma R) plays a central role in the phagocytosis of antibody complexes. We have analysed Fc gamma R (type II) RFLPs identified in TaqI- and MspI-restricted genomic DNA and found that their distribution in SLE and scleroderma did not differ significantly from controls. Hybridization with specific regions of the Fc gamma RII cDNA clone indicate that part of the Fc gamma RII alpha locus is duplicated in some individuals. A further Fc gamma RII gene has recently been identified (Fc gamma RII alpha'). This gene shows greater than 95% homology with Fc gamma RII alpha and may thus be the candidate gene for the apparent alpha duplication seen in some individuals. It is possible that an individual may possess one, two, three or four TaqI Fc gamma RII alpha/alpha' alleles, correlating with incidence and numerical heterogeneity in Fc gamma RII alpha and alpha'. The physiological effects of this numerical heterogeneity remain to be investigated.

We have isolated and deduced the structure of the beta Fc gamma RII gene from the mouse. This gene spans approximately 18 kb of DNA; the structure and nucleotide sequence has been determined and potential regulatory sites identified. This gene is composed of 10 exons that give rise to the beta 1 and beta 2 Fc gamma RII cDNA. Four exons encode the 5' untranslated region and leader sequence, one exon for each Ig-binding domain and membrane-spanning region and three exons encoding the cytoplasmic tail and 3' untranslated region. Analysis of the gene structure indicated that the beta 1Fc gamma RII and beta 2Fc gamma RII arise by differential mRNA splicing when a single 141-bp exon (exon 8) is alternately spliced to give rise to these isoforms. Sequence analysis of 2 kbp upstream of the transcription start site indicated the presence of regulatory elements, including three binding sites for the transcription factor Sp1, and Ap-4 binding site, a tandem glucocorticoid response element, and an overlapping tandem repeat. Furthermore, as in the Thy-1 gene, no CAT or TATA consensus sequences were observed. In addition, sites of methylation that regulate expression of this gene were also located at the 5' end of the gene and within several introns. A large intron located between exons 4 and 5 also contained a series of purine/pyrimidine-rich regions and a potential enhancer sequence.

A cDNA clone for an IgG-binding Fc receptor, rtFc gamma R alpha, of the rat natural killer cell line CRNK-16 is characterized here. This clone encodes an Fc gamma receptor as shown by the ability of cDNA-transfected COS cells to rosette IgG-coated sheep erythrocytes. The rtFc gamma R alpha is exceptionally homologous to the mouse moFc gamma R alpha, with 77% protein sequence identity and 71% nucleic acid identity overall. The transmembrane region of the rtFc gamma R alpha contains the sequence Leu-Phe-Ala-Val-Asp-Thr-Gly-Leu, which is present in the membrane sequences of four other Fc receptors including mouse Fc gamma R alpha, human Fc gamma RIII-2, and the Fc epsilon R alpha subunits of the rat and human high-affinity IgE-binding receptors. Also, the rtFc gamma R alpha cytoplasmic domain exhibits specific homology to other receptors derived from natural killer cells, human Fc gamma RIII-2 and mouse Fc gamma R alpha. However, the rtFc gamma R alpha cDNA clone is complementary to at least two different-sized mRNAs expressed by CRNK-16 cells, contrasting the single Fc gamma R-related mRNA species expressed by human and mouse natural killer cells. These rat mRNAs are homologous to both the 5' and the 3' end of the cDNA clone, suggesting that they may be (i) splice variants of one transcript or (ii) products of different but highly related genes.

We have recently mapped the human FCGR2 gene to chromosome 1 bands q23-q24. In situ hybridization of FCGR2 cDNA with a cell line containing a t(1:19)(q23;p13) derived from a patient with pre-B ALL has allowed a more accurate localization of this gene to chromosome 1 band q23. Furthermore, this study indicated a splitting of the FCGR2 gene or gene cluster by the t(1;19). However, Southern analysis showed no genetic rearrangement when compared with a karyotypically normal Epstein-Barr virus (EBV)-transformed cell line from the same patient. This suggests that the translocation breakpoint does not occur within the coding region of this gene.

The cDNA for the common beta Mac-1 subunit (CD11b) of the mouse LFA-1/Mac-1/p150,95 group of leukocyte cell adhesion receptors, formally designated integrin beta 2, has been cloned and sequenced. Clones were isolated from cDNA libraries made from J774 macrophage and WEHI-3B myelomonocytic tumor cells which express this subunit as a component of the macrophage activation antigen 1 (Mac-1), also known as complement receptor type 3 (CR3). This subunit is expressed as a single, abundant mRNA species approximately 2.7 kilobase (kb) in size. The 2422 base pair (bp) cDNA sequence obtained codes for a 771 amino acid protein organized with leader, extracellular, transmembrane, and cytoplamic domains of 23, 680, 23, and 46 amino acids, respectively, yielding an 82,700 mature protein of 747 amino acids. The mouse beta Mac-1 subunit is highly similar to its human counterpart with an overall sequence identity of 81% and identical positioning of 5 out of 6 potential N-linked glycosylation sites, as well as 56 Cys residues that are organized in repeating motifs characteristic of integrin beta subunits. The most highly conserved regions are the transmembrane and cytoplasmic domains where only 4 out of 69 amino acids differ, indicating that the functions associated with this domain in Mac-1-mediated processes, such as iC3b-triggered phagocytosis, have been evolutionarily conserved.

Full length cDNA clones encoding the mouse Fc gamma RI were isolated by using redundant oligonucleotide probes based on previously determined amino acid sequence of protein bound to an IgG2a antibody column. Sequence analysis of cDNA clones indicates that mouse Fc gamma RI is a transmembrane glycoprotein that is composed of three disulfide bonded extracellular Ig binding domains unlike Fc gamma RII of man and mouse. These extracellular domains contain five potential sites of N-linked glycosylation; three sites in the first domain and one in each of the second and third domains. In addition a transmembrane region is present followed by a cytoplasmic tail of 84 amino acids. Analysis of the amino acid sequence of the first two extracellular domains of Fc gamma RI indicate that these are highly homologous to the extracellular domains of Fc gamma RII; the third domain is different and shows a lower level of homology to other FcR domains but is clearly related to the Ig super-family. Transfected cells expressing Fc gamma RI were shown to bind immune complexes of rabbit IgG; and monomeric IgG2a bound to transiently transfected cells with an affinity of approximately 5 x 10(7) M-1, i.e. the receptor was of high affinity and therefore was by definition Fc gamma RI. Northern analysis demonstrated that Fc gamma RI mRNA could be detected in the Fc gamma RI+ myeloid cell lines WEH1 3B and J774. Finally, Southern analysis indicated that Fc gamma RI is likely to be encoded by a single copy gene of approximately 9 kb.

Eosinophils of approximately 95% purity were prepared from the peritoneal cavities of BALB/c male mice infected with larval cestode, Mesocestoides corti. The alloantigenic surface marker phenotype of this cell population was shown to be H-2+Ly4+Ly5+Lyt-1-,2-,3-Ly-6-,7-Ia-Thy-1-TL-. Two of four anti-Lyt-2 sera were positive when tested on purified eosinophils by using the Staphylococcus aureus protein A sheep erythrocyte rosetting method, but absorption studies indicated that this reaction was not due to anti-Lyt-2 antibodies. Eosinophils are therefore Lyt-2-, although some Lyt-2 sera contain additional eosinophil reactive antibodies. A proportion (20 to 40%) of the population of eosinophils was positive for the Fc receptor, but all were negative for the C3 receptor and for surface immunoglobulin.

The selective expression of Qa-m2 antigen on murine megakaryocyte colony-forming cells (MEG-CFC) has been demonstrated. A monoclonal antibody defining the Qa-m2 alloantigen has been shown to inhibit, in the presence of complement, in vitro formation by murine bone marrow cells of megakaryocyte colonies, but not granulocyte-macrophage, erythroid or eosinophil colonies. Inhibition of megakaryocyte colony formation was shown, by mixing and other experiments, to be due to C-mediated lysis of MEG-CFC rather than lysis of accessory cells. B-lymphocyte colony formation was reproducibly decreased by approximately 50%, demonstrating the presence of Qa-m2+ and Qa-m2- subsets of B-lymphocyte colony-forming cells (BL-CFC). MEG-CFC and granulocyte-macrophage colony-forming cells (GM-CFC) were also found to be Thy-1-, Ly-1-, Ly-6- and Ly-5-. Fractionation of Thy-1-, Ig- marrow by rosetting Qa-m2+ cells and centrifuging them through an Isopaque-Ficoll gradient yielded up to 8-fold enrichment for MEG-CFC and greater than 20-fold separation of MEG-CFC from GM-CFC. The anti-Qa-m2 antibody should be useful for further purification of MEG-CFC, and investigation of hemopoietic differentiation.

Three alloantigens encoded by Ly-6-linked genes are defined by monoclonal antibodies. The Ly-27.2 antigen is defined by antibody 5075-19.1, Ly-28.2 by 5075-3.6, -12.1, -16.10 and by 5095-16.6. The strain distribution pattern of these antibodies is the same and identical with Ly-6.2. However the tissue distribution of these antigens is unique and distinguishes these antigens from the Ly-6.2 antigen or any known antigen encoded by Ly-6-linked genes. Ly-27.2 is present on all thymocytes, T cells, and B cells but is absent from bone marrow cells, whereas Ly-28.2 is absent from most thymocytes and is present on a subpopulation of T cells and B cells but is found on 60-70% of bone marrow cells. No recombination between the Ly-6/Ly-27/Ly-28 loci was found in linkage studies using 41 recombinant inbred strains and 57 backcross mice and indicates very close linkage of these genes. In addition, close linkage to 24 minor histocompatibility genes was excluded using the Bailey HW bilineal congenic mice. The data presented indicate that either the Ly-6 complex is composed of a family of tightly linked genes or the antigens are the products of a single gene that undergoes extensive modification during differentiation.

Serological and biochemical studies using monoclonal antibodies have demonstrated that the Ly-15 cell membrane alloantigens are polymorphic sites on the lymphocyte function-associated antigen-1 (LFA-1) molecule. Ly-15.2 and LFA-1 show identical tissue distributions, being present on all thymocytes, lymphocytes, and neutrophils, and flow cytofluorometric analysis indicated identical cell surface expression of these molecules. Identity of Ly-15.2 and LFA-1 was confirmed by immunochemical analysis. The Ly-15.2 and LFA-1 molecules have an identical heterodimeric structure of Mr 180,000 alpha chain and Mr 94,000 beta chain, which coelectrophorese on two-dimensional NaDodSO4/PAGE. Furthermore, anti-Ly-15.2 and anti-LFA-1 antibodies coprecipitate the same molecule from thymocyte lysates, and peptide mapping studies show that the Ly-15.2 and LFA-1 alpha chains are identical, as are the beta chains.

The expression of Qa-2 on functional lymphocytes was investigated in vitro and in vivo by using a monoclonal anti-Qa-2 antibody. In vitro treatment of T cells with antibody and complement demonstrated that T cells mediating help or delayed-type hypersensitivity for anti-SRBC responses were Qa-2+. In addition, cytotoxic T cells and either their precursors or cells involved in their generation were Qa-2+, as were anti-HGG suppressor T cells. Panning techniques were also used to show that secondary suppressor T cells were Qa-2+ and that there may be heterogeneity in suppressor T cells defined by Qa-2 expression. In vivo treatment of mice with anti-Qa-2 resulted in decrease in immune responsiveness seen by i) prolongation of skin grafts with either H-2D or I-A differences, ii) suppression of delayed-type hypersensitivity, and iii) inhibition of T cell-mediated suppression. Finally, IgG, but not IgM, anti-body-forming cells were Qa-2+.

The effect of the H-2dm1 mutation on Qa-m2 expression was examined. The mutant strain B10.D2-H-2dm1 showed a fourfold increase in Qa-m2 expression when compared with the parental strain B10.D2. Qa-m2 molecules immunoprecipitated from B10.D2-H-2dm1, C57BL/10, and B10.D2 spleen cells were identical by two-dimensional (2-D) gel electrophoresis [isoelectric focusing (IEF) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE]). It is likely therefore that the increased Qa-m2 expression is not due to gross structural alterations of the Qa-m2 molecule; in the present study, alternative explanations are discussed.

Major structural differences occur between the thymic and peripheral T cell forms of the Ly-2/3 antigen. Thymus Ly-2/3 consists of similar amounts of two types of disulfide-linked heterodimer, alpha beta and alpha' beta (Mr alpha = 38000, Mr alpha' = 35000, Mr beta = 30000). In contrast material from peripheral T cells consists almost exclusively of alpha beta dimers. The alpha chains of thymus and peripheral T cells differ also in isoelectric point with the thymic alpha chain being the more acidic. Based on peptide mapping experiments the alpha and alpha' chains of thymus are likely to be alternatively modified forms of the same polypeptide backbone. Individual T cell clones or T cell tumors propagated in vitro exhibit either a typical thymus or a typical peripheral T cell Ly-2/3 polypeptide pattern indicating that the synthesis of both alpha and alpha' chains can occur in the same cell. The heterogeneity of thymic Ly-2/3 can be considerably reduced by removal of sialic acid residues, and after desialylation the alpha chains of thymus and a cloned cytotoxic T lymphocyte (CTL) line cannot be electrophoretically distinguished. If Ly-2 structures affected the antigen specificity of CTL, a different structural variant would be expected in individual clones. The electrophoretic identity of desialylated thymus and CTL alpha chains suggests that Ly-2 does not exhibit clonal variation in polypeptide structure and, therefore, cannot contribute to antigen specificity.

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes--Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10-20% or greater than 90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B.2 antigens are distinct from Ly-6A.2 and Ly-6D.2, however. ThB is a 16-18 kd antigen which is not associated on the cell surface with any other "Ly-6" antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.

The Ly phenotype of cells mediating skin graft rejection was determined using monoclonal anti-Lyt-1.1 and Lyt-2.1 antibodies in CBA mice that received CBA lymphoid cells from mice sensitized to C57BL/6; i.e., alloantigenic differences arising from the H-2 and non-H-2 loci. It was clear that graft rejection was due wholly to the presence of Lyt-1 cells in the inoculum and that Lyt-123 or Lyt-23 cells had no effect. Furthermore, no synergism was noted between Lyt-1 and Lyt-2 cells. In this model, both the cytotoxic T cell and cytotoxic lymphocyte precursors were shown to be Lyt-123 and these could be depleted from sensitized Lyt-1 populations that mediated graft rejection. Thus cytotoxic T cells are not responsible for skin graft rejection, but rather, this is mediated by an Lyt-1 cell. Whether this T cell is distinct from other Lyt-1 cells (T helper, T cells mediating delayed hypersensitivity) is not clear at present, but other evidence, and traditional concepts, link graft rejection and delayed type hypersensitivity as being different manifestations of the same mechanism.

The production and properties of a monoclonal anti-Lyt-1.1 antibody are described. The monoclonal antibody is of the IgG2a subclass, is cytotoxic, and reacts with Staphylococcal protein A. The tissue culture supernatant has the same activity (titer on thymus > 1/1000) as an anti-Lyt-1.1 produced by a standard immunization procedure, and after in vivo passage of the hybridoma cell line, the serum titer was > 1:10(6). The monoclonal antibody has the same strain and tissue distribution as the conventional antibody, including reactions on Lyt-1 congenic strains, and it precipitates a cell surface molecule of m.w. approximately 67,000. In nonprimed mice, there were similar numbers of Thy-1+ and Lyt-1+ cells in spleen, lymph node, and thymus. By using the monoclonal reagent, T-helper and T-killer cells in an allogenic system were shown to be Lyt-1.1.

The Fc receptor (Fc gamma R) of the murine macrophage cell line, J774, was purified by immunoaffinity chromatography then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequencing. FcR material judged to be pure by these criteria was digested with a number of enzymes to identify the cysteine residues engaged in disulfide bonds within the native structure. The results clearly establish that the mouse macrophage Fc gamma R contains two intrachain disulfide bonds, each of which connects adjacent cysteine residues within the two putative extracellular domains of the molecule. In addition, each disulfide-bonded domain was shown to contain two authentic sites of N-linked glycosylation. Extensive peptide sequencing resulted in the unexpected identification of peptide fragments from a fourth Fc gamma R whose sequences were highly homologous to sequences surrounding the two Cys residues in the amino-terminal domain of both alpha and beta 1 Fc gamma R. The fourth Fc gamma R contains a disulfide-bonded amino-terminal domain similar to beta 1 Fc gamma R.

A monoclonal antibody to the Ly-6.2 specificity, defined by strain and tissue distribution, was used to identify the cellular antigens of lymphocytes and tumor cell lines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immune precipitates demonstrated that the Ly-6.2 antigen on the surface of thymus and the T lymphoma EL-4 was a protein of 34 kd, whereas spleen cells showed two Ly-6.2 molecules of 34 kd and 56 kd. In vitro translation of EL-4 T-lymphoma poly A+ RNA followed by immunoprecipitation showed the synthesis of two Ly-6.2 precursor polypeptides. These two precursors were translated from separable mRNA molecules; the larger encoded a 54 kd polypeptide, while the second, smaller one translated a 36 kd polypeptide. Thus, the T lymphoma EL-4 contains two distinct mRNA, but only one is seen on the surface, while spleen cells contain and translate both mRNAs and both surface forms are detected. What determines the utilization of the two mRNAs and the surface expression of the two different proteins in the different tissues is not known. Whether the two mRNAs are the transcripts of one gene or arise by transcription of two separate but closely linked genes remains to be determined.

The "Qa-m9" specificity has been defined by a monoclonal antibody, and evidence for three-gene control of its expression has been demonstrated. The Qa allocation was made on the basis of H-2 linkage (H-2 congenic strains, Rl lines), differential reaction with B6.K1 and B6.K2, and biochemical analysis (two chains: 39,000 Mr and 12,000 Mr). "Qa-m9" is expressed on T and B cells in spleen and lymph node; it is present on a small population of bone marrow cells but is absent from thymocytes. The three genes involved in the expression of the antigen are: a) an H-2 linked Qa gene (possibly encoded by a gene different from Qa-2-6); b) an H-3 linked gene--probably beta 2m; and c) an H-2D linked gene that controls the amount of antigen present. It is likely that the determinant is of the "combinatorial" or conformational type, whereby the C57BL/6-type Qa heavy chain and the appropriate beta 2m light chain must both be in association. Thus, the study provides evidence for a multi-gene control of antigen expression and demonstrates the potential for generating new antigens with diversity at a post-translational level.

Human monocytes and macrophages express three different classes of cell surface receptors for the Fc portion of IgG, Fc gamma RI (CD64), Gc gamma RII (CD32), and Fc gamma RIII (CD16). We utilized a cDNA probe for Fc gamma RII to examine the modulation of Fc gamma RII mRNA by dexamethasone, a synthetic glucocorticoid, and interferon-gamma. We also determined the changes in the expression of both Fc gamma RI and Fc gamma RII protein following treatment with these agents by flow cytometry. In studies performed with the monocyte-like cell line. U937, Northern blot analysis revealed that cells treated with interferon-gamma showed a 2.5-fold increase in Fc gamma RII mRNA levels that was maximal at 14 hr and declined to 1.4-fold over baseline by 48 hr of incubation. Treatment of U937 cells with dexamethasone did not significantly change the level of Fc gamma RII transcripts, but was able to inhibit by up to 50% the increase seen following interferon-gamma treatment. The expression of Fc gamma RII protein on U937 cells was increased 56-72% after 16-24 hr of interferon-gamma treatment, but was only 18% over baseline after 48 hr of incubation. Treatment with dexamethasone caused a small, but significant, decrease in Fc gamma RII protein, and inhibited by 20-60% the induction of Fc gamma RII by interferon-gamma. The modulation by dexamethasone and interferon-gamma of Fc gamma RI protein expression on U937 cells was markedly different from that of Fc gamma RII in both magnitude and kinetics. Interferon-gamma treatment increased Fc gamma RI expression by 240% at 16 hr, and Fc gamma RI remained elevated through 48 hr. Treatment with dexamethasone decreased Fc gamma RI expression by 39%, and also inhibited by 40% the increase induced by interferon-gamma. In contrast to the findings with U937 cells, dexamethasone and/or interferon-gamma treatment had no significant effect on Fc gamma RII mRNA levels or protein expression in monocytes. However, interferon-gamma treatment increased Fc gamma RI expression on monocytes, and this increase was further augmented by treatment with dexamethasone. These data indicate that the modulation of Fc gamma RII on U937 cells is at least in part due to changes in steady state levels of Fc gamma RII mRNA. The difference between the magnitude of the changes in Fc gamma RII mRNA and protein suggests that some translational or post-translational control is involved in regulating the expression of Fc gamma RII.(ABSTRACT TRUNCATED AT 400 WORDS)

Several new aspects of the chemistry, genetics and cellular distribution of the murine Qa-2 alloantigen were apparent in an analysis of this antigen using monoclonal antibodies recognizing a Qa-2-like antigen called Qa-m2. Immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis identified the Qa-m2 alloantigen as a two-chain structure composed of a 39 000 dalton heavy chain and a 12 000 light chain which is probably beta 2-microglobulin; the heavy chain was readily distinguished from that of H-2. In addition, the expression of Qa-m2 alloantigens on the cell surface was found to be controlled by the H-2D gen with H-2Db strains carrying approximately 8-10 times greater amounts of Qa-m2 than strains carrying the H-2Dd or H-2Dq alleles. Finally, the Qa-m2 antigen, found predominantly on peripheral T cells, was present on only 10% of thymus cells. However, subpopulations of B cells (approximately 25% of all B cells) and bone marrow cells (15-20%) were also reactive. The monoclonal anti-Qa-m2 antibodies differed in their reactions from that reported for the conventional anti-Qa-2 sera, which must, therefore, be complex. The monoclonal antibodies may be useful reagents for functional analysis of T and B cell subpopulations.

The alloantigenic phenotype of 21 radiation-induced thymomas is described. Monoclonal antibodies and conventional antisera, absorbed to remove contaminating antibodies, were used to test the thymomas directly for the presence of H-2, Ia, Ly-1, Ly-2, Ly-4, Ly-5, Ly-6, Ly-9, Ly-15, Thy-1, TL, and Qa-2 antigens, and for surface immunoglobulin. The phenotypes obtained by direct tests were also confirmed by absorption studies. The tumors were all of T-cell origin (Thy-1+Ig-) and showed a restricted heterogeneity of their cell surface phenotype, concordant with the known alloantigenic phenotypes of functional T-cells. Thus the thymomas could be classified into 7 groups based on the differing expressions of Ia, Ly-1, Ly-2, Ly-4, and Ly-6 specificities; all tumors were H-2+, Ly-5+, Ly-9+, and Ly-15+. Thus a wide variety of phenotypically diverse tumors could be detected; if these represent the clonal expansion of different functional subsets, they could provide a valuable set of functional T-cells.

Immunization between CXB recombinant inbred mice selected for their identity at known loci led to the production of antisera recognizing two specificities: the previously described Ly-9.2 alloantigen and a new specificity, Ly-15.1. A monoclonal antibody to the Ly-15.2 specificity is also described. The Ly-15 specificities were found on thymus, on lymph node and spleen cells, and on liver, but were absent from kidney, brain, and red blood cells.

Human IgG Fc receptor (Fc gamma R) cDNA clones were isolated by cross-species hybridization by probing cDNA libraries with the low-affinity Fc gamma R beta 1 cDNA clone from mouse as well as a pool of oligonucleotides constructed from the nucleotide sequence of this Fc gamma R. Three cDNA clones were isolated and analysis of the predicted amino acid sequence indicated that the human Fc gamma R protein is synthesized with a 34-amino acid leader and the mature protein is composed of 281 amino acids. The extracellular region of this Fc gamma R was divided into two domains, which were very similar to each other and to the corresponding regions of both mouse alpha and beta Fc gamma Rs and showed a clear relationship to immunoglobulin variable regions. One possible N-linked glycosylation site was found in each of the extracellular domains. The human Fc gamma R leader sequence was shown to be similar to the mouse alpha Fc gamma R leader sequence, but the transmembrane region was most similar to the mouse beta 1 Fc gamma R. The intracellular domain of the human Fc gamma R was surprisingly different from both mouse Fc gamma Rs. RNA blot analysis of human cells demonstrated two transcripts (2.5 and 1.5 kilobases) that arise by use of different adenylylation signals. The cellular expression of these transcripts suggest that they encode the low-affinity p40 Fc gamma R protein.

Monoclonal anti-Lyt-1.1 alloantibody was produced as tissue culture supernatant and administered to mice. The antibody, given intraperitoneally, resulted in the suppression of all T cell functions studied, but was without direct effect on B cells. Thus, skin and tumour allograft survival was prolonged and there was suppression of the delayed-type hypersensitivity response; T cell help inthe anti-sheep red blood cell antibody response, responder cells in the mixed lymphocyte reaction (MLR), leucoagglutinin-responsive cells, cytotoxic T cell (Tc) function and the induction of Tc were either totally or partially suppressed, all these responses being mediated by Lyt-1+2- or Lyt-1+2+ cells in CBA/H mice. By contrast, there was no inhibitory effect on the MLR-stimulating or lipopolysaccharide-responsive cells. The administration of the anti-Lyt-1.1 antibody was accompanied by a depletion of Lyt-1.1+ T cells from both spleen and lymph node. These studies indicate that the monoclonal anti-Lyt-1.1 antibody is active in vivo with a selective effect on T cells. The results also have important implications for studies of T cell interactions in the mouse in vivo, and for similar studies in man.

Murine pluripotent haematopoietic stem cells have generally been assayed by their ability to form macroscopic colonies of haematopoietic cells in the spleens of heavily irradiated recipient mice (colony-forming unit-spleen or CFU-S assay). However, recent evidence suggests that there are distinct subpopulations of CFU-S. Most spleen colonies present 7-8 days after injection consist of differentiated erythroid cells, contain no primitive myeloid or erythroid precursor cells and disappear from the spleen within 3 days, whereas the majority of colonies present at 10-12 days contain primitive precursors, are multilineal and cannot be detected at 7-8 days. Furthermore, many 10-day-old spleen colonies do not contain cells capable of forming spleen colonies in secondary irradiated recipients (an index of the self-renewal capacity of stem cells) whereas at day 14 almost all colonies contain these CFU-S. These studies suggest that only when colonies are scored at or later than 11-12 days is the spleen colony technique adequate for the assay of multipotential stem cells. We now report an antigenic difference between subpopulations of CFU-S forming early (day 8) and late (day 14) spleen colonies which has been used to purify multipotential haematopoietic stem cells from murine bone marrow.

Six monoclonal anti-Ly-2.1 antibodies are described that arose from a single fusion using the spleen from a 129/ReJ mouse immunized with CBA/H thymus cells. The specificity was determined from the strain distribution, and testing of congenic strains where all six monoclonal antibodies detected the Ly-2.1 alloantigen. Furthermore, the antibodies fall into two groups: Group I (four antibodies) detected the expected number of Ly-2+ thymocytes and peripheral T cells, whereas Group II (two antibodies) detected 10% fewer peripheral T cells. Functional studies demonstrated that the Ly-2.1 determinant detected by a Group I antibody (49-31.1) was presented on cytotoxic T cells (Ly-1+2+), on concanavalin A (Con A)-induced suppressor T cells (Ly-1-2+), but absent from helper T cells (Ly-1+2-). One antibody (49-11.1) precipitated a 68,000-75,000 mol.wt glycoprotein, which on reduction yielded 30,000 and 35,000 mol.wt moieties. Thus, by functional, genetic and biochemical criteria these antibodies detected the Ly-2.1 specificity. In addition, a monoclonal, noncytotoxic, IgGl, anti-Thy-1.2 antibody is described.

A cDNA clone encoding the mouse Ly-17+ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional Fc gamma R investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG gamma 1/2b and rabbit IgG complexes.

In order to identify possible mechanisms regulating the expression of Fc gamma RII, we have examined the methylation status of the beta Fc gamma R gene in a panel of Fc gamma RII (+) and (-) cells belonging to several different lineages. We used beta 1 cDNA probes, derived from beta Fc gamma R gene transcripts which encode murine Fc gamma RII molecules. We found that all CCGG sequences detected with these probes were methylated in the genomic DNA of the Fc gamma RII-(-) cells. By contrast, two CCGG sites were found to be selectively unmethylated in the DNA of all Fc gamma RII(+) cells tested. These sites could be assigned to the region of the 5' end of the beta Fc gamma R gene. Besides, the treatment of Fc gamma RII(-) thymoma cells BW5147 with 5-azacytidine induced a hypomethylation of the beta Fc gamma R gene concomitantly with the transcription of that gene as seen by Northern blotting and the expression of functional Fc gamma RII. Conversely, the DNA-methylating agent ethyl methanesulfonate completely reversed the phenotype of the 5-azacytidine-treated cells to that of the Fc gamma RII(-) BW5147 parent cells. In ethyl methanesulfonate-treated cells, the beta Fc gamma R gene was remethylated and the corresponding transcript was no more detectable. We conclude that the methylation of a specific 5' segment of the beta Fc gamma R gene regulates the expression of Fc gamma RII in murine T cells, B cells, mast cells, and macrophages, possibly by controlling the gene transcription.

Using monoclonal antibodies, we have analysed the distribution of three recently described Qa antigenic determinants (Qa-m7, Qa-m8 and Qa-m9) on murine clonable hemopoietic progenitor cells and spleen colony-forming units (CFU-S). Cytotoxicity experiments showed that Qa-m7 was expressed on almost all the progenitor cells (colony-forming cells, CFC) of megakaryocytes (MEG-CFC), erythroid cells (E-CFC), B lymphocytes (BL-CFC), and mixed colonies (MIX-CFC) as well as day 13 CFU-S, and a major proportion of granulocyte-macrophage colony-forming cells (GM-CFC) and day 8 CFU-S. Experiments using four sources of granulocyte-macrophage colony-stimulating activity suggested differential expression of Qa-m7 on subpopulations of GM-CFC, those preferentially forming macrophage colonies having lowest Qa-m7 antigen density. Immune rosetting techniques demonstrated the selective expression of Qa-m8 on approximately 50% of MEG-CFC, MIX-CFC and day 13 CFU-S, a pattern similar to that of Qa-m2. In contrast, Qa-m9 was not detected on any of the primitive hemopoietic precursors assayed. The results demonstrate the complexity of the Qa antigenic system, and suggest a possible role for these antigens in hemopoietic differentiation.

A neutrophil-specific alloantigen (Gm-2.2) was defined by a monoclonal antibody, 5119-4/7. The Gm-2.2 antigen is found only on bone marrow neutrophils or calcium caseinate-induced neutrophils and is absent from all lymphoid cells examined as well as adherent thioglycollate-induced peritoneal exudate cells and the nonhemopoietic tissues, kidney, liver, heart, brain, and red blood cells. Furthermore, unlike mature neutrophils, granulocyte/macrophage progenitor cells are Gm-2.2-, suggesting that Gm-2.2 is a differentiation antigen for the neutrophil series. The Gm-2 locus is linked to, but distinct from, the Ly-6 locus.

The Ly-6 locus is now regarded as a gene complex consisting of at least five closely linked loci (Ly-6A-Ly-6E) whose polymorphic products are identified by monoclonal antibodies and distinguished by different tissue distributions. Ly-6 has been assigned by other investigators to chromosome (Chr) 9 (linked to Thy-1) or to Chr 2. We report that the Ly-6 gene complex, together with the Xp-14 and Gdc-1 loci, is situated on Chr 15 linked to Gpt-1. These new linkage data are derived from four sources: (1) three separate crosses that failed to demonstrate linkage of Ly-6 to either Thy-1 on Chr 9 or to any of five genes present on Chr 2; (2) the NXSM recombinant inbred strains, which suggested the linkage of Ly-6 and Xp-14 to Gpt-1 on Chr 15; (3) several Gpt-1 and Gdc-1 congenic strains that confirmed the assignment of Ly-6 and Xp-14 to Chr 15; and (4) backcrosses that further confirmed the linkage of Ly-6, Gpt-1, Gdc-1, and Xp-14, the probable gene order being Gpt-1/Ly-6-Xp-14-Gdc-1.

A monoclonal antibody defining a unique mouse neutrophil cell-surface antigen, Gm-3.2, is described. Gm-3.2 is found on all neutrophils in peritoneal exudates and in bone marrow, and is also present on macrophages activated by thioglycolate but is absent from lymphoid, kidney, liver, heart, and red cells. Gm-3.2 is a differentiation antigen of myeloid cells, as granulocyte/macrophage colony-forming cells are Gm-3.2- while mature neutrophils are Gm-3.2+. Strain distribution pattern analysis shows linkage of the Gm-3 locus to the Ly-4, B2m, H-3 complex on chromosome 2.

The problem of the structural relationship between suppressive IgG-binding factor and low-affinity receptors for the Fc portion of IgG (Fc gamma RII) has not yet been solved. In the present work we have isolated a recombinant soluble Fc gamma RII containing only the two external domains of Fc gamma RII, and analyzed its biochemical characteristics and biological activity. A cDNA encoding Fc gamma RII was mutated by the creation of a stop codon at the Lys175 codon. L cells have been transfected with this cDNA inserted into an expression vector. A cell line was obtained that secretes recombinant soluble Fc gamma RII which reacts with a monoclonal anti-Fc gamma RII antibody and binds to IgG1, IgG2a and IgG2b murine isotypes but not to IgG3 or F(ab')2 fragments of IgG2a. The secreted molecule contains two molecular species of relative molecular mass (Mr) 44,000 and 34,000-38,000 and of pI 4.5 and 6.3. They correspond to different glycosylations of a single polypeptide of Mr 19,000. After purification to homogeneity, soluble Fc gamma RII has found to suppress secondary and primary in vitro antibody responses in a dose-dependent way. The present work shows that recombinant soluble Fc gamma RII has biochemical characteristics, immunoreactivity and biological activity similar to those of suppressive IgG-binding factor.

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12,000-14,000, in contrast to initial studies which showed the relative mass to be 33,000. Using polymorphic Ly-6-specific antibodies, the 33,000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2+, Ly-6A.2- strains and a Ly-6A.2- mutant cell line BW(Thy-1-e). This clearly demonstrated that 33,000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2+ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12,000-14,000, although a minor 33,000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipitate the 12,000-14,000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12,000-14,000 molecules were only immunoprecipitated from Ly-6A.2+ cells, whereas 33,000 molecules were precipitated from both Ly-6A.2+ cells and Ly-6A.2- cells. These findings suggest that the 33,000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.