Bovine lymphocyte antigens: serological relationships with erythrocyte and spermatozoan antigens.

Bovine lymphocytotoxicity tests with 20 unabsorbed bovine blood group sera revealed extensive reactivity which in the majority of cases had no indication of blood group relationship. Six of these sera were absorbed with selected lymphocytes to produce eleven antisera of reduced specificity. Again, most of the sera had reaction patterns which could not be related to the hemolytic patterns of any known blood group antibodies. However, five comparisons involving unabsorbed antisera and two comparisons involving absorbed antisera provided statistical evidence of similarities between their lymphocytotoxic reaction patterns and the hemolytic reaction patterns of certain blood group antibodies. Several of the sera appeared to contain related cytotoxic specificities, and three such absorbed sera may have contained an anti-J specificity. All six examined monospecific isoimmune blood group antisera contained lymphocytic reactivities not related to their hemolytic specificities. Two normal sera containing naturally occurring anti-J had no cytotoxic activity. Anti-semen sera likewise were devoid of lymphocytotoxic activity.     

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.     

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.     

Production of heterologous antibodies to T-killers of mice immunized against H-2 antigens

Rabbit antisera were obtained against cytotoxic small peritoneal lymphocytes (IPEL) of CBA (H-2k) mice immune to alloantigens C57BL/6 (H-2b) and to the enriched 5-day MLC cytotoxic blast lymphocytes (MLC--CL). After appropriate absorption by cells and tissues of intact mice the cytotoxicity of the sera was lost relative to normal lymphoid cells. The absorbed anti-CPL serum inhibited, in the presence of complement, the cytotoxic effect of CPL but not that of MLC--CL on 51Cr-labeled allogeneic macrophages. This inhibition was restricted by idiotypic and strain specificity. Conversely, the absorbed anti-MLC--CL serum inhibited the cytotoxic effect of both CPL and MLC--CL of various mouse strains, irrespective of their immunologic specificity. It is supposed that the effect of the anti-CPL serum is mainly caused by antibodies againts idiotypic determinants of the killer T receptors, whereas the effect of the anti-MLC--CL serum is due to antibodies against differentiation antigens of the proliferating lymphocytes.     

Preparation and properties of an antiplasma cell serum directed against a defined plasmacytoma cell population.

Heterologous antisera were prepared against a subpopulation of MOPC-104E tumor cells obtained by centrifugation on discontinuous BSA gradients as well as against cells from the whole tumor mass. The gradient-separated cells were more effective than the cells from the whole tumor mass in eliciting antisera not only higher titer, but also with greater specificity for plasmacytoma antigens. The unabsorbed antiserum prepared against the gradient-separated plasmacytoma population was cytotoxic for murine lymphoid cells, but not for murine kidney, liver, or brain cells. After in vitro absorption with murine thymocytes and removal of anti-immunoglobulin activity by affinity chromatography, the antiserum was found to be reactive against plasmacytoma cells, but was no longer cytotoxic for murine thymus or unstimulated spleen cells. This absorbed antiserum was also cytotoxic for LPS-, but not PHA- or Con A-stimulated normal murine spleen cells.     

Studies on the antibody composition and neutralizing activity of tetanus antitoxin sera from various species of animals in relation to the antigenic substructure of the tetanus toxin molecule

On the basis of the antigenic substructure of tetanus neurotoxin, the antitoxin compositions of horse, rabbit and human tetanus antitoxin sera, in terms of their contents of antibodies against four antigenic determinant groups (alpha, beta-1, beta-2 and the "topographic" determinant group gamma) so far known for the toxin were studied by quantitative precipitation reactions using purified toxin, complementary fragments alpha, beta and fragment beta-1 (a subfragment of fragment beta) of the toxin. The antitoxin antibody composition varied slightly depending on the antiserum preparation. In addition, different patterns of antitoxin antibody composition and toxin-neutralizing ability, characteristic of horse, rabbit and man were found: horse antitoxin sera contained all four kinds of antibodies and horse anti-gamma showed low toxin-neutralizing ability, while human antisera lacked anti-alpha and had anti-gamma with high neutralizing activity but contained anti-beta-1 with no detectable neutralizing activity. Rabbit sera showed an intermediate pattern between those of horse and human sera. In all antisera, antibodies against determinants on the isolated fragment beta account for approximately 80-50 percent of the total precipitable antibodies and anti-beta-2 antibody was invariably present. Immunodiffusion analyses showed that the antitoxin compositions of mouse and guinea pig antisera resembled those of human antisera. In mice, fragment beta was almost as efficient as whole toxin toxoid in eliciting a protective immune response on an equal weight basis, whereas fragments beta-1 and alpha were both relatively poor antigens.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines fromHLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelatedHLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Characterization of an antiserum specific for cell surface antigens of rat mast cells.

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.     

Immunological and Other Biological Characteristics of Pentons of Human Adenoviruses

Comparative hemagglutination-enhancement (HE) tests demonstrated diversified patterns of antigenic specificities both in the fiber and vertex capsomer part of pentons of human adenovirus types 3, 11 (subgroup I), 9, 15 (II), 1, 2, 4, 5, 6 (III), and 12. All fibers contained a type-specific antigen. Subgroup II and III fibers, in addition, contained specificities both unique for each subgroup and also common to the two subgroups. Fibers of serotypes 4 and 12 displayed a somewhat deviating behavior. All vertex capsomers tested shared a group-specific part. This was the only antigenic specificity demonstrable for serotype 12. Maximal penton HE titers of all sera were reached in tests with incomplete hemagglutinin of type 11. In addition, maximal HE activity of sera against individual serotypes also was recorded against pentons of other members of the same subgroup. Antigen characteristics of vertex capsomers of type 4 indicated a closer relationship to subgroup I than to subgroup III. The toxin activity of pentons was more sensitive to trypsin treatment than their capacity to function as incomplete hemagglutinin. Homotypic antipenton sera, unabsorbed or absorbed with homotypic fibers to remove antibodies against this component, and, to a varying extent, also heterotypic antipenton sera could neutralize toxin activity. Antifiber sera could neutralize toxin activity of pentons carrying short fibers (10 nm, type 3) but not of those carrying long fibers (28 to 31 nm, type 2). It is concluded that toxin activity is carried by a specific part of vertex capsomers and that cell detachment can be brought about via a direct contact between this component and cell membranes. Fiber-mediated attachment does not seem to be necessary for this biological activity to become expressed.     

Differences in the capacity of HLA sera to react with T- and B-lymphocyte populations]

A study was made of perculiarities attending the reaction of the HLA-sera with the T- and B-lymphocytes isolated from human blood. Lymphocytes were separated by removal of one of the cell subpopulation. T-lymphocytes were separated by the method of rosette-formation with sheep erythrocytes, with subsequent gradient density centrifugation. B-lymphocytes were separated similarly with the aid of rosette-formation with allogenous Rh-positive erythrocytes sensitized with Rh-sera with incomplete antibodies, and also sorption of B-lymphocytes on synthetic fiber. The cytotoxic activity of HLA-sera decreased after the removal of B-cells. But removal of T-lymphocytes was not accompanied by any reduction in the lymphocytotoxic activity. It is suggested that B-lymphocytes contained on their surface more HLA determinants than T-lymphocytes.     

Localization of antigens on the inner-acrosomal membrane of the rabbit sperm by immunofluorescence

The acrosome-reacted spermatozoa interact with the zona pellucida through their limiting inner acrosomal membrane (IAM). The antigenic properties of IAM were determined using the antibody to IAM raised in a male guinea pig. The antisera was incubated with the acetone powder of rabbit lung, liver, kidney, heart, muscle, and blood cells. The unabsorbed antibodies specifically interacted with antigens on the IAM as determined by the immunofluorescence technique.     

Maternal serum reactivity to species-specific antigens in sheep-goat interspecific pregnancy.

Three models were used to test the hypothesis that interspecific pregnancy failure between the sheep and goat is due to a species-specific, maternal antibody response. Interspecific pregnancies were established in ewes and does, sheep in equilibrium goat chimeric conceptuses produced by injection of ovine blastocysts were transferred to ovine recipients, and ovine and caprine pregnancies were established in interspecific chimeras. Complement-mediated lymphocytotoxic and hemolytic assays were used to monitor onset and titer of antibodies. Sera from 3 of 8 injection-chimera recipients reacted with all caprine peripheral blood lymphocytes (PBL) and red blood cells (RBC) tested (n = 18). Sera from 3 of 6 ewes and 7 of 7 does also were pancytotoxic to PBL of the other species (n greater than or equal to 20). Absorptions with xenogeneic RBC generally removed the reactivity. The data were consistent with responses to species-specific, monomorphic antigens expressed on PBL and RBC, and probably trophoblast. The response preceded or coincided with interspecific pregnancy failure in does, but not in ewes. Accordingly, no xenoreactivity was observed in chimera sera but caprine pregnancies were resorbed (n = 16) and ovine pregnancies developed to term (n = 11). The data did not support the hypothesis that failure of caprine pregnancy in ewes or chimeras is due to a species-specific, maternal antibody response. In contrast, a maternal, cytotoxic antibody response to species-specific antigen(s) may contribute to failure of hybrid or ovine pregnancy in does.     

Seasonal variation of bovine anti-J in the tropics

Close to the Equator at an altitude of 1750 m above sea level a total of 27 cows of various breeds have been studied with regard to the fluctuation of their anti-J naturally occurring antibodies. Over 16 consecutive months the average total score of anti-J varied from a minimum of 20 in October to a maximum of 37 at the end of December. Practically all the sera had the lowest level of anti-J in October. This is about two months after the lowest yearly maximum temperature. It is concluded that gradual changes in temperature can cause variation in anti-J titer, and that the magnitude of the seasonal temperature changes influences the fluctuation of the natural anti-J.     

Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3162 = 4.8 % ) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand.
Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1 %) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida.
Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.     

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.     

Immunological detection of heterogeneous O-antigen containing lipopolysaccharides in Escherichia coli

The components of the cell envelopes of Escherichia coli O1:K1, O7:K1, O18:K1 and O83:K1 strains were separated on SDS-polyacrylamide gels. Longitudinal slices (50 microns thick) of the gel were incubated with typing sera for E. coli O1, O7, O18 and O83, followed by detection of the bound antibodies with 125I-labelled protein A and autoradiography. The antisera reacted with many cell envelope components of strains both with the homologous O-serotype and heterologous O-serotypes. With O-typing sera cross-reactions with heterologous cells and cells boiled for 2 h were found. Up to 40 serotype-specific bands at regular positions with molecular weights between 12000 and 100000 were demonstrated. Since these bands were also observed when purified lipopolysaccharide and unabsorbed homologous O-typing sera were used, it was concluded that these bands represented lipopolysaccharide molecules with increasing molecular weight, all of which contained O-antigen specific immunodeterminants. The band patterns were not influenced by the growth conditions of the cells or the various isolation procedures for the cell envelopes. Comparison of various strains serotyped as O18 revealed strain differences with respect to their lipopolysaccharide band patterns. In the case of O21- and O83-serotyped strains lipopolysaccharide cross-reactions, which were detected by agglutination, were analysed in detail using the gel immunoradioassay method. These cross-reactions appeared to be caused by the presence of common determinants on their lipopolysaccharides and polysaccharide-like material. The cross-reacting antibodies could be removed by cross-absorption. It is concluded that the immunological detection of lipopolysaccharides and other components of E. coli in gels is an important tool in (1) the control of the specificity of typing antisera, (2) the study of the nature of cross-reacting antigens and (3) the study of the nature and uniformity of the various O- and K-serotypes.     

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3/62 = 4.8%) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand. Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1%) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida. Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.     

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H^MAN SERUM

That portion of horse and human serum globulin precipitated by 33 per cent saturation with ammonium sulfate and precipitated on subsequent dialysis was taken as euglobulin; and the fraction precipitated between 33 and 50 per cent saturation and remaining in solution on subsequent dialysis was taken as pseudoglobulin. The sera of rabbits injected with either of these antigens gave precipitation with both. However, two distinct and fraction-specific antibodies could be demonstrated by absorbing the sera with the one antigen, and testing the supernatant fluid with the other. The experimental results are adequately explained on the basis that there are at least two antigenically distinct globulins in serum which we may term globulin I and globulin II and which are largely associated with so called euglobulin and pseudoglobulin respectively. The ordinary methods of salting out and dialysis do not effect complete separation and each globulin preparation contains a trace of the other antigen. The antisera to these euglobulin and pseudoglobulin preparations therefore contain antibodies to both antigens. Each protein solution precipitates all the antibody specific for the one antigen and in addition, by virtue of the trace of contaminating protein, precipitates a portion, and only a portion of the antibody specific for the other antigen. The fact that antisera to whole serum contain these same fraction-specific antibodies suggests that this immunological specificity is an inherent property of two globulins present as such in serum and is not an artifact induced by their precipitation and purification. Lipoids extracted from the globulins by ether, petroleum ether, and alcohol give no demonstrable reaction with antisera to these globulins; antisera absorbed with a large excess of lipoid are not affected as regards their reactivity with the original protein; and globulins extracted with ether and petroleum ether at room temperature are not affected as regards their reactivity with antisera. It is concluded that the immunological specificity of the globulin fractions as evidenced by the precipitation reaction is not determined by lipoids associated with the protein.