Monoclonal antibody differentiates chicken A system alloantigens

A monoclonal antibody (ISU-cA) was produced that recognized certain alloantigens of the chicken A blood group locus. Antigens produced by alleles A3, A4 and A8 were positive, and those produced by A2 and A5 were negative, by haemagglutination. The specificity of ISU-cA for chicken A blood group antigens was demonstrated by serologic analyses, genetic crosses and competitive inhibition of binding by anti-A alloantisera. To our knowledge, this is the first reported monoclonal antibody against a chicken blood group alloantigen system other than the B complex.     

Ontogeny and expression of chicken A blood group antigens

Expression of chicken red blood cell (RBC) surface antigens was studied by using a monoclonal antibody (ISU-cA) specific for chicken A blood group antigens. Erythrocytes were examined from embryos of 3-18 days of incubation and from chicks at hatch up to 21 weeks of age. Specific antigens were detected on embryonic RBC surfaces by immunofluorescence as early as 3 days of incubation. Antigenic expression was examined by both haemagglutination and immunofluorescence and found to increase with age from embryos to mature birds. The antigen concentration on the cell surface was found to be affected by genotype; heterozygotes had an intermediate level of antigen between that of the two parental genotypes. These data confirm the co-dominance that is observed with most blood group antigens. Flow cytometric analysis allowed confirmation that the entire erythrocyte population gradually increased in antigenic expression over time, rather than having an antigen-negative subpopulation being replaced by a positive subpopulation.     

Characterization of two distinct disulfide-linked B-G molecules in the chicken

Alloantisera specific for B-G antigens recognized a complex of molecules of apparent molecular weights of 90 and 98 Kd under nonreducing conditions and molecules of 40, 44, and 48 Kd under reducing conditions on both embryo- and adult-derived peripheral red blood cells (RBC). The chicken B-G molecules produced a unique two-dimensional "diagonal" pattern. Two antisera permitted the characterization of the complex B-G molecular profile as a homodimer composed of 48-Kd subunits and as a heterodimer composed of 40- and 44-Kd subunits. A rabbit antiserum produced against B-G molecules preferentially recognized the 48-Kd reduced molecules, suggesting that the 90-Kd molecule was a homodimer composed of two 48-Kd molecules. One B-G reagent was capable of recognizing only the 98-Kd nonreduced B-G molecule that gave rise to 40- and 44-Kd molecules under reducing conditions, suggesting that the 98-Kd molecule was a heterodimer composed of 44- and 40-Kd subunits. Adult chicken B-G2 molecules produced a variety of two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) patterns depending on the characteristics of the reagent employed in the immunoprecipitation. B-G molecules were immunoprecipitated from primitive and definitive chicken RBCs but not from any nonerythroid cells tested. B-G molecules were not expressed by avian erythroblastosis virus (AEV)-transformed erythroleukemia cells, nor were they induced to appear with butyric acid-induced erythroid differentiation.     

Evidence for two populations of B-L (Ia-Like) molecules encoded by the chicken MHC

Two specific alloantisera detecting B-L (Ia-like) antigens on chicken lymphocytes of the B ⁶ and B ¹⁵ haplotypes were found to cross-react strongly. Anti-B-L6 and anti-B-L15 alloantisera both reacted with B-L molecules on B6 and B15 lymphocytes as demonstrated by immunofluorescence and SDS-PAGE analysis of ¹²⁵I-labeled B-L antigens isolated by incubation with anti-B-L alloantisera. Absorption studies showed that the anti-B-L alloantisera reacted with at least two kinds of antigenic determinant, one set shared by B-L6 and B-L15 molecules and another set specific for each haplotype. In spite of the absence of genetic evidence for more than one B-L locus in the chicken B complex, it was shown by sequential antibody incubations that these two different B-L antigenic determinants are associated with at least two separate species of B-L molecules, indicating the presence of at least two B-L loci within the MHC of the chicken.     

Specificity of H-2 antigens expressed on mouse blastocysts

A highly sensitive cytotoxicity assay was used to detect H-2 antigens on mouse blastocyst stage embryos of the b, a, k, and d haplotypes. The assay was based on the principle that live embryos incorporate 3H-thymidine into DNA whereas embryos killed with antiserum and complement do not. The use of specific alloantisera showed that blastocysts of different haplotypes express different H-2 antigens. Thus, positive evidence was obtained for the expression of Kd and Dk molecules and negative evidence for the expression of Db, Kk, and Dd molecules. Evidence was also obtained that blastocysts express different H-2 antigens than those found on adult lymphocytes. Unexpected cross-reactions were found when some of the alloantisera were tested on blastocysts of different haplotypes. It is proposed that the aberrant expression of H-2 antigens on embryos might facilitate their escape from surveillance by the maternal immune system.     

Interspecies cross-reactivity of bovine lymphocytotoxic sera with pig lymphocytes

Forty-three bovine BoLA antisera were tested on pig lymphocytes by a microlymphocytotoxicity test. Twenty-five were found to be cytolytic. Fifteen sera detected the A blood group antigen on porcine lymphocytes but showed no reaction with the J antigen on bovine lymphocytes. Six BoLA reagents reacted with all pig cells tested. Cross-reactions with SLA antigens were observed in only four sera, the highest correlation being recorded with SLA-W7 (r = 0.87). Bovine alloantisera are not of value for SLA typing.     

Immunochemical variants of HLA-B27

Detailed study of HLA-B27 was prompted by the extremely strong associations between this antigen and spondyloarthropathies. Despite the relative homogeneity of this antigen when defined by alloantisera, B27 reactivity with the monoclonal antibody B27M2 suggests previously unrecognized heterogeneity. To define and confirm this heterogeneity on a molecular level, detergent extracts were prepared from B cell lines derived from individuals reactive (+) or unreactive (-) with the B27M2 antibody. Extracts were immunoprecipitated by specific allogeneic or monoclonal antibodies and analyzed by two-dimensional polyacrylamide gel electrophoresis. By this method the B27M2+ and B27M2- variants of HLA-B27 had different isoelectric points (pl) and could be distinguished from each other and from a different (Bw44) control alloantigen. Blockade of glycosylation by pretreatment of cells with tunicamycin did not alter pl but did reduce HLA antigens by approximately 3000 daltons. These data demonstrate that B27 antigens can be subdivided into subsets with different molecular composition. The effects of this heterogeneity upon the associations of B27 and disease are not yet known.     

Detection of at least two distinct mouse I-E antigen molecules by the use of a monoclonal antibody

In an attempt to determine whether the expression of more than a single Ia antigen is determined by the I-E subregion of the mouse major histocompatibility complex (MHC), sequential immunoprecipitation analyses were performed by using a monoclonal antibody and alloantisera reactive with I-E subregion products. 3H-leucine-labeled glycoprotein preparations obtained from H-2d spleen cells were precleared with the monoclonal antibody 14-4-4S and then examined for residual Ia activity precipitable by an alloantiserum detected by SDS-polyacrylamide gel electrophoresis. Residual Ia activity was observed for all three strains of the H-2d haplotype tested. The residual Ia activity could be detected by sera absorbed with B10.A spleen cells, indicating that products of the I-E subregion rather than of the I-C subregion were responsible for this activity. No separable I-Ek molecules were detected in products of B10.A cells with the use of combinations of two monoclonal antibodies (including 14-4-4S) and several appropriate alloantisera. These findings indicate the presence of at least two similar but distinct I-E antigens encoded by the H-2d haplotype.     

Evidence for an IgD homologue on chicken lymphocytes

Chicken lymphocyte membrane immunoglobulins (Ig), were precipitated with mouse monoclonal antibodies specific for heavy and light chain isotypes and analyzed by polyacrylamide gel electrophoresis. Very little or no membrane-bound IgG and IgA was detected. After sequential precipitation and removal of IgM reactive with any of three monoclonal anti-mu antibodies, anti-light chain antibody precipitated residual Ig with a relative electrophoretic mobility similar to that of IgM. Under reducing conditions, these surface Ig molecules had a heavy chain that appeared slightly larger (approximately 81,000 daltons) than mu-chain (approximately 79,000 daltons), and light chains of approximately 25,000 daltons. Complete clearance of membrane-bound IgM reactive with an anti-mu allotype antiserum left similar molecules precipitate by monoclonal anti-light chain antibody. These non-IgM molecules could be detected on the surface of lymphocytes from blood, spleen, bursa and the B cell line RAV-1, but not from thymus or blood from an agammaglobulinemic chicken. After capping of B cell surface IgM with anti-mu, immunofluorescent staining with anti-light chain antibody revealed residual Ig molecules disturbed across the surface of more than 90% of the IgM-bearing cells. The data suggest the existence of an avian homologue of mammalian IgD. Affinity-purified goat anti-mu antibodies and a fourth monoclonal anti-mu antibody reacted with both IgM and the putative IgD molecules, which suggests that the IgD homologue shares at least one common determinant with chicken IgM.     

Identification of the MT3 molecule using two-dimensional gel electrophoresis and alloantisera

The MT3 antigen is defined serologically as a DR supertypic specificity and is strongly associated with DR4, DR7, and DRw9. To determine whether the MT3 molecule is distinct from the DR molecule, DR4 and MT3 antigens were immunoprecipitated from 125I-labeled plasma membrane glycoproteins of a DR4-homozygous, MT3-homozygous B lymphoid cell line, Wa, and compared by two-dimensional (2-D) gel electrophoresis. The precipitates with two different anti-DR4 alloantisera and with three different mouse antibodies against human Ia monomorphic determinants gave the same 2-D gel pattern consisting of one heavy chain with a molecular weight of 34 000 and a set of light chains with a molecular weight of 30 000, indicating that these polypeptides are the components of the DR4 molecule. On the other hand, all three anti-MT3 alloantisera used precipitated an identical set of anti-MT3 alloantisera specific light chains with a molecular weight of 30 000, and one heavy chain with a molecular weight of 34 000. The pI of the MT3 light chain was more acidic than that of the DR4 light chain. The amount of MT3 light chains was much smaller than that of DR4 light chains in unlabeled plasma membrane glycoproteins. Thus, we have demonstrated directly using 2-D gel electrophoresis and anti-MT3 alloantisera that the MT3 antigen is a new human Ia molecule distinct from DR4.     

At least two loci encode polymorphic class I MHC antigens in the horse

Summary. Six monoclonal antibodies and ten alloantisera were used to precipitate cell surface molecules of approximately 44kDa (class I MHC antigens) from radiolabelled equine peripheral blood lymphocytes. All ten antisera were raised against antigens of a single donor horse (horse 0834, ELA-A2,-A2). Four methods of producing antisera were compared: one or two pregnancies, skin allografting, and skin grafting followed by pregnancy. Immunization by pregnancy appeared to produce antibodies against class I products only, while skin grafting raised antibodies to class II antigens as well. Nine of the antisera were raised across an entire MHC haplotype barrier, while one recipient carried the ELA-A2 antigen of the donor. The pregnancy antiserum raised across this barrier probably identifies a second polymorphic class I locus in the horse. Sequential immunoprecipitation using this antiserum in the first stage and an anti-MHC haplotype antiserum or monoclonal antibody reagent in the second stage supported this hypothesis. Gene products of this second ELA class I locus are immunogenic in pregnancy.     

Biochemical evidence that equine leucocyte antigens W13, W22 and W23 are present on horse major histocompatibility complex class II molecules

A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens precipitated from horse lymphocyte lysates using (cross-reacting) antibodies to human class I and class II MHC molecules confirmed the results obtained.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence microcytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Distinct classes of human T-cell activation antigens

The characterization of three groups of antigens expressed by activated human T lymphocytes and detected by monoclonal antibodies is reported. Antigens defined by OKT19, OKT21, and OKT22 do not appear on in vitro activated T cells until increases in DNA synthesis become apparent and are not detected on most Interleukin 2 (IL-2)-independent cell lines and normal peripheral blood lymphocytes, monocytes, and granulocytes. Cell surface molecules reactive with the monoclonal antibodies OKT23 and OKT24 are displayed prior to any notable increase in DNA synthesis and are present on IL-2 independent cell lines, irrespective of lineage. T23 and T24 do not appear on peripheral blood cells and their distribution more closely resembles that of the T9 antigen (the receptor for transferrin) than antigens of the other groups. The third group of antigens, T14 and T20, have been classified as "early" antigens relative to DNA synthesis. They are expressed by distinct populations of normal lymphoid cells as well as by some IL-2-independent cell lines. Display of each group of activation antigens on T lymphocytes can be induced by either phytohemagglutinin, purified protein derivative from tuberculin, or allogeneic non-T cells, is not restricted to the OKT4+ or OKT8+ subsets, and is predominant on cells exhibiting the light-scattering properties of blast cells. The relative lack of expression of these antigens among normal peripheral blood cells make them attractive candidates for identifying changes in the status of immune activation.     

Inhibition of the induction of cytolytic T lymphocytes with alloantisera directed against H-2K and H-2D gene products.

Alloantisera directed at gene products of the H-2Kd or H-2Dd loci on the stimulator cell were shown to inhibit specifically the generation of cytolytic T lymphocytes to those antigens. Thus, masking the antigens of one H-2 locus on the stimulator cell inhibits the induction of CTL to products of that locus but does not inhibit the induction of CTL to antigens of another H-2 locus. Alloantisera inhibition of the induction of cytolytic T lymphocytes occurred with both normal and primed responder cells and also occurred when the stimulating antigens were on whole cells or purified plasma membrane. Absorption on the appropriate spleen cells removed the inhibitory capacity of these alloantisera.     

Heterogeneity of human melanoma-associated antigens defined by monoclonal antibodies and conventional xenoantisera

Summary Immunochemical analysis of cultured human melanoma cell detergent extracts and spent culture medium with conventional xenoantisera and monoclonal antibodies identified four types of 94,000 (94K) dalton molecules and two types of high-molecular-weight melanoma-associated antigens by the following characteristics: (1) association with other components, (2) mobility in SDS-PAGE under reducing and nonreducing conditions, (3) antigenicity, and (4) presence in spent culture medium. Conventional xenoantisera were found to contain antibody populations to antigenically distinct structures, some of which have similar apparent molecular weights. Immunodepletion studies showed that the antigenic determinant detected by the monoclonal antibody 225.28S to a high-molecular-weight melanoma-associated antigen was expressed on a subpopulation of the antigens defined by the conventional xenoantiserum #8995. These data prove that antibodies reactive with antigens of similar molecular weight cannot be assumed to identify the same structures, and indicate that tumor-associated antigens may be heterogeneous in the expression of antigenic determinants defined by monoclonal antibodies.Visiting investigator from the Veterans Administration Hospital, Minneapolis, MinnesotaVisiting investigator from Sapporo Medical College (Japan). Abbreviations used: MAA, melanoma-associated antigen; PBS, phosphate-buffered saline; NP40, nonidet P40; MoAb, monoclonal antibody; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 2-ME, 2-mercaptoethanol     

Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for Lyt-2/3 function

The lysis by allospecific cytolytic T lymphocytes (CTL) of the BALB/c lymphoma ST-4.5, a cell line that can be induced by interferon-gamma (IFN-gamma) to express increased amounts of major histocompatibility complex (MHC) class I antigens, was investigated. Culture of ST-4.5 in IFN-gamma increased the surface expression of Kd molecules from originally low levels and Dd from undetectable amounts by approximately fivefold as determined by fluorescence-activated cell sorter (FACS) analysis, whereas the levels of several other antigens (Ld, I-Ad, Thy-1, Lyt-2, L3T4, and LFA-1) were not affected. The lysis of ST-4.5 by Dd- and Ld-specific CTL clones correlated with the expression of those antigens on target cells as determined by both FACS and biochemical analysis. Lysis of ST-4.5 by CTL clones specific for Kd antigen fell into two distinct groups: those that could lyse targets cultured either normally or in IFN-gamma, and those that could only lyse targets that had been precultured in IFN-gamma. The apparent sensitivity to antigen exhibited by the Kd-specific CTL clones predicted their sensitivity to inhibition of target lysis by anti-Lyt-2/3 antibody. Those CTL clones that were only active against ST-4.5 expressing higher amounts of surface antigen (resulting from IFN-gamma preculture) were readily inhibited by anti-Lyt-2/3 antibody, whereas those CTL capable of lysing normally cultured targets having lower amounts of surface antigen were heterogeneous in their sensitivity to anti-Lyt-2/3; some were inhibitable, whereas others were resistant. In addition, another CTL clone that was resistant to inhibition by anti-Lyt-2/3 alone was readily inhibited by a synergistic combination of anti-Lyt-2/3 plus anti-Kd (but not anti-Dd or Ld) antibodies. These results indicate that CTL antigen receptor sensitivity to (or affinity for) antigen and the level of specific antigen expression by the target cell may both be important criteria in assessing Lyt-2/3 molecule function in CTL-mediated cytolysis. The function of recognition-associated molecules such as Lyt-2/3 may be to strengthen and increase the number of receptor-ligand binding events that facilitate CTL-target membrane interactions that lead to the lysis of the target cell.     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

Lymphocyte alloantigens of the horse. III. ELY-2.1: a lymphocyte alloantigen not coded for by the MHC

A new polymorphic locus of the horse which has several unusual properties is described. The suggested name for the locus is ELY-2 . The gene product of one allele at this locus, designated ELY-2.1 , has been identified with antisera raised as a result of pregnancy. Antibody to ELY-2.1 was first detected on day 55 after conception in the serum of a mare in first pregnancy. This early onset of antibody is similar to that seen for antibody to ELA antigens, and suggests that the source of the antigenic stimulus may be the tissue of the equine endometrial cups.
The antisera identifying ELY-2.1 are cytotoxic and kill all peripheral blood lymphocytes from horses carrying the antigen. ELY-2.1 is a cell surface molecule expressed on lymphocytes, erythrocytes, and platelets. Other cell types have not been investigated. The overall phenotypic frequency of ELY-2.1 in several horse breeds was 16 %. The ELY-2.1 antigen is controlled by an autosomal, dominant gene which is not coded by the ELA region (the major histocompatibility complex of the horse), nor is it identical to the ELY-1 locus, which codes for another cell surface alloantigen of equine lymphocytes. Stimulator cells carrying ELY-2.1 did not induce proliferation of ELY-2.1 negative responder cells in mixed cultures of horse lymphocytes. Attempts to raise alloantisera to other alleles of the ELY-2 locus through immunization with lymphocytes were unsuccessful. It is possible that the alternate allele(s) does not code for a gene product which is expressed. The function and biochemical nature of the ELY-2.1 molecule are unknown.