GENETIC AND BIOCHEMICAL CHARACTERIZATION OF ANTIGENS ENCODED BY THE LY-24 (Pgp-1) LOCUS1

Three allele-specific monoclonal antibodies to Pgp-1 (Ly-24) were used to biochemically characterize the cell surface structures with which they reacted and to map the gene(s) coding for these antigens. The targets of these three monoclonal antibodies (mAb) were shown to be encoded by a gene situated on chromosome 2 close to β₂m [gene order (Pgp-1-β₂m-a)] and no recombination between the loci detected by the three antibodies was revealed by genetic analysis. The genetic mapping of loci and tissue distribution of these antigens suggested that they might all correspond to a particular allelic form of the mouse phagocyte glycoprotein-1 (Pgp-1) antigen. Biochemical and serological analysis confirmed that this was indeed the case and revealed that all three mAbs were directed to one epitope. It is surprising that the tissue distribution defined by one mAb (Ly-24A) was different from that for the two other (Ly-24B) antibodies, despite the serological and biochemical identity of their respective targets. The possible reason for this unusual finding is discussed.     

The cell surface phenotype of mouse neutrophils

Using monoclonal antibodies, mouse peritoneal neutrophils were typed for the presence of 23 cell surface alloantigens, the expression of which was quantitated by flow cytofluorometry and compared with that of lymphocytes. The H-2K and H-2D alloantigens and beta 2-microglobulin were present on all neutrophils, but Ia and Qa antigens were not detected. It was found that Ly-5.1, Ly-15.2, Ly-21.2, Ly-24.2 (Pgp-1) and Ly-25.1 were present on greater than 90% of neutropils; Ly-6.2 and Ly-27.2 were absent, but Ly-28.2 (encoded by an Ly-6 linked gene), was present on greater than 90% of neutrophils. As expected, the lymphocyte-specific antigens Ly-1.1, Ly-2.2, Ly-3.1, Ly-7.2, Ly-12.1 and Thy-1.2 were absent from the neutrophils. When compared with lymphocytes, marked differences in alloantigen expression on neutrophils were seen for Ly-5.1, Ly-24.2 and Ly-28.2. These studies should be of value in the study of neutrophil structure and function.     

A monoclonal antibody to Ly-6 gene product inhibits generation of functionally active T cells and recognizes single antigenic specificity whose expression is up-regulated in virus-transformed rat fibroblast.

In order to elucidate the relationship between the structure and function of proteins encoded for by the Ly-6 gene complex, a cDNA was constructed for a Ly-6.2 specificity and then monoclonal antibodies (mAb) generated to bacterially synthesized protein. The addition of one of these mAb, designated Pb-19, inhibited the proliferative response of T cells to concanavalin A (Con A) or major histocompatability complex (MHC) alloantigens. Reactivity of Pb-19 to the Ly-6 specificity was blocked by a known anti-Ly-6.A.2 mAb but not by an anti-Ly-6.E.1 mAb. This mAb detected a Ly-6.A.2 specificity (a 33,000 MW antigen) whose expression was increased in a transformed rat fibroblast containing the entire genome of bovine papillomavirus.     

Amplification of the immune response by agonistic antibodies

During the last seven years, more than 1000 munine monoclonal antibodies (mAb) have been made which react with human leucocyte differentiation antigens. International workshops have extensively evaluated these mAb; over 25 differentiation antigens have been identified and included in an international WHO-approved nomenclature^1–3, called clusters of differentiation (CD). These are identified by both serological and biochemical analyses. In this article Ed Clark and Jeff Ledbetter summarize a subset of these mAb specific for differentiation antigens that, as agonists, induce activation, growth, or differentiation of lymphocytes.     

Restricted usage of VH and V kappa genes by murine monoclonal antibodies against 3-fucosyllactosamine

We are studying the structure and regulation of murine antibodies against the 3-fucosyllactosamine antigenic determinant. Analysis of the sequences of seven BALB/c IgM, kappa monoclonal antibodies (mAb), obtained from four fusions, indicates that these antibodies exhibit restriction in their usage of VH and VL genes. Based on a combination of mRNA sequences and Southern filter hybridization data, all seven light chains are encoded by V kappa 24B and J kappa 1 gene segments. Complete mRNA sequences of the heavy chains revealed that all seven mAb are encoded by VH441, six antibodies are encoded by JH4 and one uses a JH3 gene segment. The VH441 gene segment and all seven mAb contain a potential glycosylation site at Asn 58 in complementarity-determining region (CDR)2. In contrast to the similarity of the VH regions, the heavy chain CDR3 segments exhibit considerable heterogeneity. They are encoded by three D segments, they vary in length from 7-9 amino acids and display differences in their deduced amino acid sequences. The VH441 gene segment also encodes antibodies against four other carbohydrate antigens, levan, galactan, dextran and galactosyl globoside. The use of a single gene segment to encode antibodies against five different antigens suggests that the domain encoded by VH441 might be particularly well adapted for forming sites that bind carbohydrate determinants. Glycosylation of CDR2 might contribute to the unique properties of this VH domain.     

Intracellular events associated with inhibition of B cell activation by monoclonal antibodies to HLA class II antigens

We have investigated several aspects of the inhibitory effects of monoclonal antibodies (mAb) directed against MHC class II antigens in B cell activation/proliferation, using a panel of mAb specifically reactive with antigens encoded by HLA class II loci (DP, DQ, DR). All mAb except the anti-DP mAb inhibited significantly anti-mu plus B cell growth factor-induced DNA synthesis. Only one mAb, however, which was reactive with gene products of all three class II loci (DP, DQ, DR) inhibited anti-mu-induced DNA synthesis as well as c-myc mRNA expression. In addition, the same mAb inhibited the early events induced by anti-mu stimulation alone, including phosphatidylinositol turnover and elevation of [Ca2+]i. In contrast to previous findings in the murine system, none of the anti-MHC class II mAb used in this study increased the cAMP levels.     

Elevated expression of Pgp-1 (Ly-24) by murine peritoneal B lymphocytes

B cell expression of the surface glycoprotein, Pgp-1 (Ly-24), was evaluated using flow cytometric analysis. Pgp-1 expression on naive, conventional (splenic) B cells was low but could be increased by mitogenic stimulation. Pgp-1 expression on naive peritoneal B cells was higher than expression by unmanipulated conventionalB cells, suggesting the possibility that peritoneal B cells have been activated in vivo. However, the elevated expression of Pgp-1 by peritoneal B cells was not accompanied by increased expression of surface Ia but was correlated with Ly-1 status. Further, Pgp-1 expression by B cells from germ-free mice did not differ from that of normal animals. The elevated expression of Pgp-1 by peritoneal B cellsis superficially similar to that provoked by mitogenic stimulation of conventional B cells; however, several characteristics suggest that the elevated expression of Pgp-1 by peritoneal B cells does not reflect prior activation of these cells.     

Pro-T cells in fetal thymus express c-kit and RAG-2 but do not rearrange the gene encoding the T cell receptor β chain

Ten percent of 15-day fetal thymocytes of mice were Pgp-1⁺Thy-1^lo cells. Half were strongly stained with monoclonal antibodies (mAb) recognizing the oncogene product, c-kit, but were not stained with mAb against non-T cell markers such as B220, Mac-1 and Gr-1. The isolated Pgp-1⁺c-kit⁺ thymocytes showed no rearranged bands for V-DJ and D-J of T cell receptor (TcR) β, but Pgp-1^?-c-kit^? thymocytes showed D-J rearranged bands. Both cells expressed the RAG-2 gene which is required for the V(D)J recombination process. When Pgp-1⁺c-kit⁺ thymocytes were cultured in 2-deoxyguanosine-treated alymphocytic fetal thymus, they became TcR-expressing mature type T cells, but this differentiation was reduced by the addition of anti c-kit mAb. These data indicate that Pgp-1⁺c-kit⁺ thymocytes are pro-T cells with the potential to differentiate mature T cells in the thymic environment. This study also indicates that c-kit-mediated signals promote the differentiation of thymocytes during their early stages.     

Definition of selectable cell surface markers for human chromosomes and chromosome segments in rodent-human hybrids

A panel of 22 monoclonal antibodies (MAbs) recognizing 21 distinct human cell surface antigens was tested by mixed hemadsorption assays for reactivity with a large number of rodent-human somatic cell hybrids containing different subsets of the human chromosome complement. The serological typing results permit the assignment of six gene loci determining cell surface antigens to human chromosomes 3, 6, 11, 19, 20, and 22. In addition, analysis of hybrids retaining deleted copies (but no normal homologs) of specific human chromosomes provides regional assignments for 18 gene loci, located on seven different chromosomes. These findings extend and refine the genetic map for human cell surface antigens and identify new selectable markers for defined chromosome segments.     

Polymorphism of Qa and Tla loci of the mouse

Congenic lines carrying H-2 haplotypes derived from wild mice were typed serologically with polyclonal and monoclonal antibodies specific for Tla, Qa-1, and Qa-2 antigens. The typing revealed the presence of a minimum of five Tla, four Qa-1 , and three Qa-2 alleles in the 32 lines. Two new Tla , two new Qa-1 , and one new Qa-2 alleles could be described. This polymorphism of Tla and Qa loci is lower than that detected at the K and D loci in the same lines. The serological typing for Qa-2 antigens correlates remarkably well with previously published results of typing with cytolytic T lymphocytes. This correlation supports the identity of loci detected by the two methods.     

Analysis of the NIH workshop monoclonal antibodies to human melanoma antigens

Reagents exchanged at the 2nd workshop on monoclonal antibodies (MoAb) to human melanoma antigens were analyzed using both serological and immunochemical assays. The analysis by laboratories participating in the workshop of our MoAb 225.28S, 345.134S, 376.96S, 465.12S, and 763.24TS reaffirms our own analysis of these reagents in that (1) they all react with the majority of melanoma cell lines tested and (2) the reactivity of MoAb 225.28S and 763.24TS is much more restricted than that of MoAb 345.134S 376.96S, and 465.12S. Our serological analysis revealed that the majority of workshop reagents reacted with cultured melanoma cells. Immunochemical analysis of these monoclonal antibodies allowed for their division into three groups according to the molecular weights of the antigens recognized in immunoprecipitation experiments, greater than 100 kd, 80-100 kd, and DR antigens. Further analysis of the first two groups of monoclonal antibodies by immunodepletion and antibody binding inhibition assays revealed that MoAb 9.2.27, 225.28S, and 763.24TS recognize distinct determinants with a heterogeneous distribution on subpopulations of a high molecular weight melanoma associated antigen. MoAb 376.96S and 705.F6 recognize either the same or spatially close determinant(s).     

Monoclonal antibodies to the murine Ly-2.1 cell surface antigen.

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.     

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.     

Characterization of a 95,000 molecule on sheep leucocytes homologous to murine Pgp-1 and human CD44.

The phagocyte glycoprotein-1 (Pgp-1) antigen of mice is a 94,000 MW molecule with a wide tissue distribution, but no attributed function. We produced a monoclonal antibody (mAb) termed 25-32 which recognizes the Pgp-1 molecule of numerous mammalian species, including humans and sheep. Preclearing experiments with I42/5, a rat anti-mouse Pgp-1 mAb that cross-reacts with human Pgp-1, established the specificity of 25-32 for human and sheep Pgp-1. Moreover, an antibody recognizing human CD44, termed F10-44-2, also reacted with the same molecule as that recognized by 25-32 and I42/5, so establishing the co-identity of CD44 and Pgp-1. Within the sheep thymus, Pgp-1 was expressed most strongly by medullary thymocytes and stromal cells, and by small numbers of cells at the subcapsular cortex. Pgp-1 was expressed early in thymic ontogeny; all 35-40-day gestation fetal sheep thymocytes were intensely Pgp-1+, but by 80 days the number of reactive thymocytes had decreased to adult levels. The expression of Pgp-1 on lymphocytes was markedly increased after stimulation with mitogens, or with phorbol esters and ionomycin. The highly conserved nature of Pgp-1 through evolution, its expression on virtually all cell types within the body, and its increased expression on rapidly dividing cells indicate that this molecule mediates an important function, possibly serving as a hormone or metabolite receptor.     

Murine monoclonal antibodies and human alloantisera specific for HLA inhibit monocyte phagocytosis of anti-D-sensitized human red blood cells

Evidence is presented that monoclonal antibodies (mAb) against some human major histocompatibility complex (MHC) gene products and human sera containing HLA antibodies strongly inhibit immune phagocytosis of anti-D-sensitized red blood cells by human monocytes. w6.32HL, a mAb against a monomorphic class I antigen of high cell surface density, revealed the strongest inhibition among mAb reactive with MHC class I products. mAb with preferential reactivity for monomorphic and polymorphic DR and DQ epitopes (L203, L227, IIIE3, Tü22, Genox 3.53 and IV12) were noninhibitory. Definite inhibition was also apparent with a mAb against DRw52/MT2 (I-LR2) and with an antibody to class II antigens of high cell surface density (2MC3). Human sera containing HLA antibodies showed strong inhibition of immune phagocytosis up to a dilution of 1/1000. This inhibition could not be abrogated by platelet absorption. This indicates that human sera inhibiting immune phagocytosis may comprise at least two types of antibodies: cytotoxic HLA-specific antibodies which may or may not be inhibitory, and inhibitory antibodies against monocytic antigens not necessarily cytotoxic. These latter antibodies may recognize HLA DR or another as yet undefined gene product within, or closely associated to, the human MHC.     

Antigenic phenotyping of isolated and in situ rodent follicular dendritic cells (FDC) with emphasis on the ultrastructural demonstration of Ia antigens

The antigenic phenotype of mouse lymph node follicular dendritic cells (FDCs) was studied by immunocytochemical techniques. Indirect fluorescence was used in conjunction with monoclonal antibodies to localize FDC surface antigens on FDC-enriched cell preparations and in cryostat sections. Lymph nodes from rats and mice were also labeled directly for Ia antigens with fluorescein- or peroxidase-conjugated Ia-specific monoclonal antibodies (i.e., MRC Ox4 and 10-2.16, respectively). Lymphoid tissue was also prepared for electron microscopy to allow clear distinction between Ia antigens of B lymphocytes and FDCs in situ. In these experiments, gold-labeled antigen was used to clearly identify FDCs and their processes among the Ia-positive cells of lymph node follicles. The labeling observed by light and electron microscopy showed that FDCs expressed Ia in situ and in vitro. Additional surface determinants shown to be expressed by FDCs included H2-K, common leukocyte antigen, and the receptor for the Fc portion of IgG1 and IgG2b. Neither macrophage antigens, such as Mac-1, Mac-2, Mac-3, and F4/80, nor the lymphocyte markers Ly-1, Ly-2, and Thy-1 were expressed by FDCs. Thus, the antigenic phenotype of FDCs, along with their distinctive dendritic morphology, their nonphagocytic and nonadherent nature, and their ability to trap and retain immune complexes on their plasma membrane, identifies them as a unique cell population.     

Distinct HLA-DR epitopes and distinct families of HLA-DR molecules defined by 15 monoclonal antibodies (mAb) either anti-DR or allo-anti-Iak crossreacting with human DR molecule. I. Cross-inhibition studies of mAb cell surface fixation and differential binding of mAb to detergent-solubilized HLA molecules immobilized to a solid phase by a first mAb

A series of HLA-DR-reactive mouse anti-human B cell or anti-Ia monoclonal antibodies (mAb) have been used to explore the serological complexity of human class II antigens at the determinant level, using two techniques: (a) cross antibody-binding competitor assays using ¹²⁵I-labeled and -unlabeled mAb were performed to study the topological organization of the corresponding determinants to determine epitopic clusters recognized by this collection of mAb and (b) differential reactivity of mAb to detergent-solobilized solid-phase-immobilized HLA-DR molecules to determine epitopes expressed on identical DR isotypes. The fifteen mAb could be classified according to the first technique as falling into three different epitopic clusters. Using the second technique, we were able to define at least two independent molecular subsets, one co-expressing two of the three epitopic clusters and the second expressing only the third one. We could not formally identify molecular subsets expressing only one of the first two clusters, using the second technique. The precise serological mapping of the determinants recognized by various anticlass II mAb should prove very useful if such mAb were to be introduced in anticlass II-specific T cell clone blocking experiments. We anticipate that some of them should facilitate the correlation at the clonal level between the T cell repertoire and the epitopes or molecular subsets defined by these mAb. However, within mAb belonging apparently to a same cluster, some could mediate different biological effects.     

Relationship of B cell Fc receptors to T cell recognition of Mls antigen

The Mls locus on chromosome 1 controls the expression of cellular determinants that are responsible for stimulating mixed lymphocyte reactions between H-2-identical strains. However, the biochemical nature of Mls antigenic determinants remains undefined. It has been proposed that Ly-17 lymphoid cell surface antigens (also known as Ly.m.20.2 and LyM-1) and Mls antigens could be identical because they are both encoded by loci on chromosome 1 and display similar tissue distribution. The Ly-17 locus encodes polymorphic alleles of the IgG Fc receptor (Fc gamma R). In the present study, two approaches were used to address the question of whether Fc gamma R are involved in T cell recognition of Mls antigen. In the first approach we tested the effect of Fc gamma R blockade by heat-aggregated mouse IgG or anti-Fc gamma R monoclonal antibodies (2.4.G2) on the ability of an Ia+, Fc gamma R+ Mlsa-expressing B cell hybrid (LBB.3.4.16) to stimulate interleukin 2 secretion by an anti-Mlsa-specific T cell hybrid. We show that blockade of Fc gamma R does not inhibit the Mlsa-specific stimulation of T cells during a 24-h culture period in which Fc gamma R remain blocked. In the second approach, we derived irradiation-induced variants of LBB.3.4.16 to dissociate Fc gamma R expression and Mls antigen expression. We describe 2 LBB variants which no longer stimulate Mlsa-reactive T cells but do express Fc gamma R. Compared to parental LBB cells, the capacity of variant LBB cells to present soluble antigen to Ia-restricted T cells is unaffected. Collectively, these results indicate that Fc gamma R expression and Mlsa antigen stimulation can be dissociated. We conclude that Fc gamma R expression may be necessary, but not sufficient for T cell recognition of Mls antigen.     

Expression of extra class I major histocompatibility antigens on T-cell acute lymphoblastic leukemia (ALL) lymphoblasts

The presence of extra HLA antigens has been demonstrated, serologically and biochemically, on the surface of lymphoblasts from a patient with acute lymphoblastic leukemia of the T-cell subtype (T-ALL). Family analysis of this patient revealed the presence of the expected antigens, plus an additional HLA antigen (A24) which could be demonstrated by cytotoxicity on the lymphoblasts. Absorption studies revealed that the lymphoblasts had the ability to remove cytotoxic antibodies from alloantisera; similarly, absorption of these alloantisera with normal cells removed the reaction against the extra antigen from the lymphoblasts. The extra HLA molecules were also demonstrated by one-dimensional IEF. Two heavy chain-like molecules, together with the beta 2m subunit, were obtained after removal of appropriate antigens from externally labeled leukemia cells by the use of monoclonal antibody W6/32, which detects a class I specific determinant. The pI of the one heavy chain was shown to be very similar to that of the serologically detected A24. Our data thus suggest that the extra antigens detected by serological reagents may have been due to activation of silent class I MHC gene(s) at the protein level.