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.     

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.     

A human alloantiserum precipitates Ia-like molecules from chronic lymphocytic leukemia cells.

Alloantigens specific for human B lymphocytes can be identified with selected antisera. These antigens have similarities to murine Ia antigens in that they are found on human B lymphocytes and are controlled by genes linked to genes controlling HLA. Chronic lymphocytic leukemia cells bearing B cell antigens were labeled with 3H leucine and the membrane components reacting with the B cell antisera isolated by immunoprecipitation. These membrane components had m.w. of 33,000 and 24,000 daltons similar to the murine Ia antigens. The results complete the homology of murine Ia and human B cell alloantigens.     

Rabbit antisera to human B cell alloantigens: effects on the mixed lymphocyte response.

Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes.Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′)₂ or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.     

CD8 is needed for development of cytotoxic T cells but not helper T cells.

A mutant mouse strain without CD8 (Lyt-2 and Lyt-3) expression on the cell surface has been generated by disrupting the Lyt-2 gene using embryonic stem cell technology. In these mice, CD8+ T lymphocytes are not present in peripheral lymphoid organs, but the CD4+ T lymphocyte population seems to be unaltered. Cytotoxic response of T lymphocytes from these mice against alloantigens and viral antigens is dramatically decreased. Proliferative response against alloantigens and in vivo help to B lymphocytes, however, are not affected. These data suggest that CD8 is necessary for the maturation and positive selection of class I MHC restricted cytotoxic T lymphocytes but is not required on any of the intermediate thymocyte populations (CD8+CD4-TcR- or CD4+CD8+TcRlow) during the development of functional class II MHC restricted helper T cells.     

Evidence of a second polymorphic ELA class I (ELA-B) locus and gene order for three loci of the equine major histocompatibility complex

Two antisera, B-442 and R-2046, were produced by immunizing offspring with purified peripheral blood lymphocytes from a parent matched for the ELA-A specificity carried on the unshared haplotype. Absorption analysis demonstrated that these antisera contained at least two families of cytotoxic antibodies, one directed against antigens present on T and B cells, and a second directed preferentially against antigens present on surface Ig positive cells. Immunoprecipitation studies using these antisera demonstrated that both antisera contain antibodies specific for glycoproteins with molecular weights characteristic of class I and class II MHC antigens. In lymphocyte typing tests of unfractionated lymphocytes, only the class I activity was readily detectable since the class II activity killed less than 25% of the cells. Family studies demonstrated that these antisera recognize products of genes linked to the ELA system. Based on two recombinants in an extended family it became apparent that the specificities detected by B-442 and R-2046 are not products of the ELA-A locus, but rather they are products of at least one other locus, defined in this paper as ELA-B. In this family a third recombinant was found between the A blood group system and the ELA-A locus. Based on these three recombinants, the most probable linear relationship of the following genes is: A blood group system/ELA-A/ELA-B.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Cyclosporine-induced tolerance in experimental organ transplantation. Evidence of diminished donor-specific cytotoxicity relative to donor-specific proliferative response

Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules.     

High titres of antibody to murine leukaemia virus in lymphocyte (Ly) alloantisera

The radioimmune precipitation (RIP) assay was used to examine the antibody titres against endogenous AKR murine leukaemia virus (MuLV) in a number of antisera to lymphocyte (Ly) alloantigens. The sera from normal donor and unimmunized recipient mice used in raising the alloantisera were also examined for anti-MuLV activity. It was found that all the antisera had high anti-MuLV titres and that in all but one case alloantigen immunization augmented the anti-viral titres. The degree of augmentation did not appear to be related to the anti-MuLV titre in the donor strain sera. Three I-region antisera were also examined for anti-MuLV antibodies and were found to have lower anti-viral titres than the Ly antisera even though immunization to I-region products greatly augmented the anti-viral titre. These results caution against the use of Ly antisera in characterizing the phenotype of lymphoid tumour cells without prior virus absorption.     

High titres of antibody to murine leukaemia virus in lymphocyte (Ly) alloantisera

The radioimmune precipitation (RIP) assay was used to examine the antibody titres against endogenous AKR murine leukaemia virus (MuLV) in a number of antisera to lymphocyte (Ly) alloantigens. The sera from normal donor and unimmunized recipient mice used in raising the alloantisera were also examined for anti-MuLV activity. It was found that all the antisera had high anti-MuLV titres and that in all but one case alloantigen immunization augmented the anti-viral titres. The degree of augmentation did not appear to be related to the anti-MuLV titre in the donor strain sera. Three I-region antisera were also examined for anti-MuLV antibodies and were found to have lower anti-viral titres than the Ly antisera even though immunization to I-region products greatly augmented the anti-viral titre. These results caution against the use of Ly antisera in characterizing the phenotype of lymphoid tumour cells without prior virus absorption.     

Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25-27 April 1984

The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25-27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Antiserum inhibition of the mixed lymphocyte culture (MLC) interaction. Inhibitory effect of antibodies reactive with HLA-D-associated determinants.

Two antisera, procured by immunization within HLA-A- and HLA-B-identical and HLA-D-incompatible unrelated combinations, were cytotoxic to B lymphocytes from the immunizing donor and from persons sharing his HLA-D-incompatible phenotype(s). The sera strongly and specifically inhibited lymphocytes from these donors when used as stimulating cells in mixed lymphocyte culture (MLC) reactions, while specific responding cell inhibition was less evident. The inhibitory effect was retained in the immunoglobulin G (IgG) and the F (ab)′₂ fractions of these antisera. Inhibition was observed when the antisera were added within 48 hr after initiating the MLC. We conclude that these antisera contain antibodies reactive with structures closely associated with HLA-D determinants and that these may be human analogs of the mouse Ia antigens.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.     

Identification of new murine lymphocyte alloantigens: antisera prepared between C57L, 129 and related strains define new loci

Antisera prepared by immunizing between the strains 129 and C57L and other related strains identified new antigens expressed on lymphocytes and in particular on thymocytes. Absorption analysis demonstrated that the antisera were complex, and contained several new antibodies including some which were not cytotoxic, but could be detected by rosetting. The loci defined by these antibodies are referred to as Ly-9, Ly-11, Ly-12, Ly-13, and Ly-14, although several of the antigens were not confined to lymphocytes. In addition, the Ea-7 specificities, previously considered to be purely red-cell alloantigens, were also found on thymocytes.     

The presence of Ia-like determinants on a subpopulation of human T lymphocytes

By planned immunization within HLA-A-, and -B-compatible and HLA-D-disparate combinations, we have raised two antisera which are cytotoxic in complement-dependent cytotoxicity (CDC) tests with B lymphocytes, but not with T lymphocytes, from the immunizing donor and other donors sharing the immunizing HLA-D phenotype. The sera were found previously to inhibit the stimulating capacity of cells in MLC and the Fc receptor of cells producing EA rosettes, suggesting that they may detect alloantigens analogous to Ia antigens in mice. Although apparently non reactive with T cells in CDC tests and immunofluorescence, these sera were investigated further for their potential interference with some T-cell functions. After pretreatment with the appropriate antiserum and complement, the cells behaved normally as responding cells in mixed lymphocyte culture, as precursors to the cytotoxic cells in cell-mediated lympholysis, and as cells responding to the purified protein derivative (PPD). However, the response to phytohemagglutinin (PHA) was reduced at low concentrations of this mitogen, and the response to concanavalin A was strongly reduced at all concentrations, indicating that some subpopulations of human T cells also carry Ia-like specificities.     

Recognition of antigens by T lymphocytes

Summary The present review briefly summarizes our knowledge of antigen-specific B and T lymphocyte receptors. Antigen-specific receptors on mammalian B lymphocytes are mainly monomeric IgM and IgD consisting of conventional immunoglobulin heavy and light chains. The nature of the T lymphocyte receptor which can specifically recognize antigens is not yet fully defined. However, it seems that conventional light chains do not participate in the build up of this receptor, and that the receptor is made up of heavy chains of a new immunoglobulin class which has to be further characterized and which we call Tau-chain. The variable region of the T lymphocyte receptors share idiotypic determinants with the corresponding B lymphocyte receptors. The possible linkage between the T cell idiotypes present on the Tau-chains and molecules which are under the control of genes of the Major Histocompatibility Complex of the species are discussed.In the last part of the review two methods for the induction of specific transplantation tolerance in adult animals are described. These methods are based on the concept that T lymphocytes reactive against alloantigens bear idiotypic determinants against which a specific auto-immune response can be initiated.     

Distribution of alloantigens on human Fc receptor-bearing lymphocytes: the presence of B cell alloantigens on sIg-positive but not sIg-negative lymphocytes.

Human peripheral blood lymphocyte subpopulations were analyzed for the presence of B cell alloantigens with a microcytotoxicity assay. B cell alloantigens were found exclusively on sIg-positive lymphocytes and were not present on sIg-negative, Fc receptor-bearing lymphocytes or sIg-negative, Fc receptor-negative T lymphocytes.     

Serologically detected lymphocyte antigens in Holstein cattle1

Lymphocytes from 103 Holstein cattle were tested with 11 antilymphocytotoxic sera. Four of these sera were produced by whole blood immunization; these generally yielded lymphocytotoxicity against a large number of animals in each test panel. The majority of the sera were collected from parous cows which had never been immunized. Observations about this latter group of sera are (1) lymphocyte antibodies may occur by alloimmunization in foetal-maternal interaction, (2) normal sera from non-immunized parous cows generally had a narrower specificity of antibodies than from parous cows immunized with blood from unrelated animals and in some cases these antisera may be monospecific, and (3) these sera from parous cows are easy to obtain without the need for laborious immunizations. Analysis of these lymphocytotoxic sera indicate several reagents were obtained which possessed different antibodies; these were useful in detecting polymorphism of cattle lymphocyte antigens. Using these 11 sera, an analysis of the reactions patterns among 103 Holsteins, including parent-offspring data resulted in the postulation of several alleles. These data suggest that cattle lymphocyte antigens are very polymorphic and inherited.     

Serologically detected lymphocyte antigens in Holstein cattle

Lymphocytes from 103 Holstein cattle were tested with 11 antilymphocytotoxic sera. Four of these sera were produced by whole blood immunization; these generally yielded lymphocytotoxicity against a large number of animals in each test panel. The majority of the sera were collected from parous cows which had never been immunized. Observations about this latter group of sera are (1) lymphocyte antibodies, may occur by alloimmunization in foetal-maternal interaction, (2) normal sera from non-immunized parous cows generally had a narrower specificity of antibodies than from parous cows immunized with blood from unrelated animals and in some cases these antisera may be monospecific, and (3) these sera from parous cows are easy to obtain without the need for laborious immunizations. Analysis of these lymphocytotoxic sera indicate several reagents were obtained which possessed different antibodies; these were useful in detecting polymorphism of cattle lymphocyte antigens. Using these 11 sera, an analysis of the reactions patterns among 103 Holsteins, including parent-offspring data resulted in the postulation of several alleles. These data suggest that cattle lymphocyte antigens are very polymorphic and inherited.