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Two Hypoxanthine/Aminopterin/Thymidine-sensitive cell sublines (L142 and L231) have been derived from independent lymphoblastoid cell lines of B lineage. After propagation for more than 100 population doublings (1 year) in culture, these cells still retained a doubling time between 19 to 20 hours, near diploïdy and relatively low (L142) and high (L231) secretion rate of M immunoglobulins. Near diploid hybrid cells were easily generated with leukocytes from the spleen, the gut lamina propria or the mesenteric lymph nodes of pigs immunized against the transmissible gastroenteritis virus. Both the tumor sublines and the B cells were derived from histocompatible miniature pigs (d/d haplotype). Demonstration of fusion between the tumor sublines and B-cells was supported by the selection of hybridomas making the antigen-specific heavy (alpha isotype) and light chains from the B cell parent as well as the heavy and light chains of the lymphoblastoid parent. Moreover, some hybridomas were found to secrete only class A (dimeric) or class M immunoglobulins (0.2-10 micrograms/ml). Forty hybridomas secreted antibodies reactive in a virus-enzyme-linked cell immunoassay against cell-bound antigens and two were found to produce an antibody active only against the infected cell monolayer. Construction of intraspecies hybridoma can be used to perpetuate lymphocyte subsets useful for the study of the porcine immune system.

A lymphoblastoid cell line (B1) was isolated in culture following a brief exposure to 5-azacytidine from peripheral-blood mononuclear cells of a boar previously injected with cells (Shimozuma) producing porcine retrovirus (Tsukuba-1) and suffering a severe non-neoplastic syndrome at autopsy. B1 cell line and 5 of its sublines were propagated for more than 100 generations, retaining doubling times comprised between 16.8 and 27.5 hr and growing readily in agarose or agar (plating efficiency: 5 to 50%). Karyotype analyses showed that 4 sublines were nearly diploid, except for cells of L14, which displayed a monosomy affecting chromosome 18 pair. Two sublines (L35 and L45) were considered as being of T-cell lineage, since MSA, antigen was observed on the surface of approximately 30% of cells. Three sublines (L23, L14 and L52) were considered of B-cell lineage, since membrane immunoglobulins were observed on the cell surface. In addition, sublines L23 and L52 were actively secreting immunoglobulin of mu isotype. Retrovirus particles were evidenced in gradient-purified preparation of 200-fold-concentrated cell culture supernatants of the B1 cell line, L14, L35 and L52 sublines, using both a reverse transcriptase activity assay and electron microscopic observation. These cell lines can be used to select for porcine retrovirus variants with transforming potential for lymphocytes of B and T lineages.

A method is described for the purification of sheep lymphocytes carrying class II MHC antigens. After incubation of purified blood lymphocytes on anti-IgM-coated petri dishes, the adherent fraction contained 95% sIg-positive cells determined by immunofluorescence. When tested with cross-reacting anti-class II (bovine and human) monoclonal antibodies, more than 95% of these cells were positive either by immunofluorescence or cytotoxicity. This technique will permit studies of the polymorphism of sheep class II antigens.

Antibody-dependent cellular-cytotoxicity (ADCC) was studied by the 51chromium release test after a 18 h incubation of non-immune porcine blood leukocytes with labelled chicken erythrocytes sensitized with pig antibodies. It was shown that ADCC was inhibited by incubation of leukocytes with aggregated immunoglobulins. In our assay, pig lymphocytes were shown to be inactive whereas non-lymphocyte pig leukocytes are effector cells even at low effector:target ratios.

Immunization of sheep or horse with pig, canin or horse thymocytes produces heteroagglutinating antibodies, which allow to define two heterophile antigens:--the first one, HC, immunogen for sheep, is localized on pig, human and canin thymocytes, as well as on red blood cells of the two latter species;--the other, HCP, immunogen for horses, is situated on the red blood cells and thymocytes of the same three species. HC is distributed on various cells and in similar fashion in pig and human, except for the pig red blood cells.

The immunoglobulins obtained from a sheep anti-pig thymocyte serum were made specific for a subpopulation of porcine lymphocytes by adsorptions with pig red blood cells and bone marrow cells. In the presence of rabbit complement, these immunoglobulins killed a porcine lymphocyte subpopulation similar to the subpopulation of lymphocytes forming rosettes with sheep red blood cell (E-rosettes) when enhanced by dextran. Without complement, these immunoglobulins inhibited the E-rosette phenomenon at a dilution near to the cytotoxicity titre of the serum, but did not inhibit the rosettes formed with activated zymosan (ZC rosettes). These two different tests revealed the presence of antibodies directed against pig T-lymphocyte specific antigens and allowed the quantitication of T lymphocytes in different organs.

The use of alpha-naphthyl acetate esterase (ANAE) as a T cell marker in some other species and the broad correlation of incidence of ANAE-positivity and E rosette-formation in the pig suggest that ANAE-staining may be a T-cell marker in the pig. However, by studying the staining of lymphocytes within a variety of rosettes in fixed preparations a similar incidence of pig blood lymphocytes were found to be ANAE+ among T cells (E rosettes formed in dextran), B cells (antiglobulin rosettes) and Fc-gamma receptor-bearing B and T cells (EA rosettes in saline and dextran): complement (C') receptor-bearing cells showed a higher incidence of staining than other lymphocytes. Analysis of staining morphology suggested that certain morphologies within the B and T lineages may be confined to subpopulations. Thus ANAE positivity is certainly not a marker identifying blood T lymphocytes but could be of some value indicating subpopulations of B and T lymphocytes.

An efficient placental barrier protects the pig fetus from all exogenous antigenic stimulation. The results of studies on different subpopulations of lymphocytes, histological localization, and stimulation as well as immunization in utero with allogeneic lymphocytes have confirmed that the pig fetus becomes immunocompetent at around 80 days of fetal life (total length of gestation: 115 days). Because the newborn piglet has had no "immunological experience" in utero, it can only elaborate a primary response which is often advantageous to pathogenic microorganisms. Under natural conditions, immune protection is provided by the mother in the form of colostrum. This colostrum provides systemic immunity and is followed by milk which insures local immunity in the piglet gastrointestinal tract. However, the great disadvantage of maternal antibodies is that they suppress the active synthesis of new antibodies in young pig. Thus, the beginning of active immunity in the piglet is a function of the ratio of maternal antibody level to the level of antigenic stimulation.

Pulmonic and peripheral blood lymphocyte subpopulations in healthy and Mycoplasma suipneumoniae-infected pigs were compared. The T- and the B-lymphocyte populations were counted, the B cells by the complement receptor (zymosan-complement rosettes) technique and the T cells by the ED-rosette technique (sheep RBC-dextran rosettes). The T cells were found to predominate among pulmonic, as well as blood, lymphocytes. Pulmonic B-cell and blood T-cell percentages were increased after mycoplasma respiratory tract infection. However, blood B-cell and pulmonic T-cell percentages were not significantly affected. A significant (P less than 0.001) correlation between pulmonic and blood T-lymphocyte compositions was found; conversely, no correlation was observed between blood and pulmonic B-cell percentages. These data could imply that pulmonic B cells are predominantly involved in local immune reactions after a mycoplasma respiratory tract infection.

Qualitative and quantitative changes in pig lymphocyte sub-populations (T, B, L and nulls) were studied in the mammary gland of primiparous sows, as well as the appearance and development of plasma cells in relation to the 3 phases of mammary development: gestational, colostral and lacteal phases. Results showed: 1) An early colonisation of mammary epithelium from the beginning of the gestation phase by the three T, B and L lymphocyte subpopulations; 2) The presence of activated T lymphocytes, especially during the colostral phase; 3) The appearance of plasma cells during the colostral phase and their increase during the lacteal phase; 4) Not only the T, B and L lymphocyte subpopulations increased during the lactation but there was also a special increase in null lymphocyte subpopulation. These results give new data which are particularly important in allowing us to envisage immunization schemes for the pregnant sows, with the aim of enhancing local mammary or lactogenic immunity by transfer of antibodies and lymphocytes to the newborns.

Lymphocyte subpopulations derived from different pig organs (blood, spleen, mesenteric lymph nodes) were separated by a simple, rapid and cheap panning technique, using either normal or ozone treated Petri dishes with bovine serum albumin. Slg+ lymphocytes could thus be obtained with a purity of up to 95% by adhesion onto Corning plastic dishes. The purified cells retained their proliferative activity with regard to lectins. The subpopulation including PT4 and PT8 was then separated by another panning on ozone-treated plastic dishes with a purity of 80-90%.

Conditioned taste aversion induced by either an immunosuppressive (cyclophosphamide) or non immunosuppressive (lithium chloride) drug reduced expression of a T cell mediated immune response that is highly susceptible to stress hormones. These results demonstrate that the proposed concept of conditioned immunosuppression really represents another example of how stress can alter normal regulation of cell mediated immune events.

An intriguing and important biological question is how the immune system is subject to regulation by exogenous factors. In the mouse system, our laboratory has demonstrated that a wide variety of environmental stimuli, such as heat, cold, short-term restraint and conditioned-taste aversions, affect cell-mediated immune events in vivo. The reduction in cell-mediated immunity that is caused by restraint can be abolished by adrenalectomy or treatment with an inhibitor of corticosterone biosynthesis. We have therefore examined the effect of a short-term restraint stressor on the immune system of pigs. Five-to-eight-week-old-pigs were restrained by placing them in an expanded-metal box for 2 h each day for 3 consecutive days. Restraint caused a significant increase in serum cortisol. Preliminary evidence indicates that restraint significantly reduced size of the thymus gland from 0.14% to 0.10% of body weight and also significantly reduced in vivo phytohemagglutinin-stimulated, cell-mediated reactions by 25%. Addition of cortisol at a concentration of 130 ng/ml to phytohemagglutinin and concanavalin A-stimulated porcine lymphocytes in vitro reduced mitogenesis of splenocytes, peripheral blood lymphocytes and thymocytes. Although these data reflect changes in immune function caused by an acute environmental stressor in vivo and by cortisol in vitro, research from another laboratory indicates that longer-term restriction of pigs with tethers and farrowing crates can also decrease immune events.(ABSTRACT TRUNCATED AT 250 WORDS)

Study of the distribution of P+S+ and P-S+ cells in various lymphoid organs of pig revealed that the thymus contains the highest proportion of P+S+ cells (75.5 +/- 4.2%). In vivo treatment of neonatal piglets with cortisone: (i) Reduced both the area of the thymic cortex even more than the medulla and the P+S+ cells more than the P-S+ cells, suggesting the respective origin of these two populations in these two tissue compartments. Moreover, cortisone-resistant thymocytes responded better than the control untreated pig thymocytes to Con A and PHA stimulation. (ii) Reduced the number of P+S+ cells in peripheral organs. Separation of adult thymocytes into P+S+ and P-S+ cell fractions showed that P-S+ cells responded better than P+S+ to Con A and PHA stimulation. Cortisone-resistant thymocytes and P-S+ cells showed the same cell size distribution and both are enriched in medium sized cells. Thus, it is concluded that the property of rosette formation with pig red blood cells allows the identification of two subsets of pig thymocytes with different biological properties and thymic locations, which by analogy with other species may prove to have different patterns of emigration to peripheral organs.

A panel of cytotoxic monoclonal antibodies (Moab) derived from mice immunized with porcine thymocytes has been developed which reacts with monomorphic determinants on porcine peripheral blood lymphocytes (PBL). These Moab recognized from 25% to 100% of PBL as tested by flow microfluorimetry One of the Moab (PT 81) that bound 39% of PBL, 30% of splenocytes and 65% of thymocytes as determined by flow microfluorimetry was selected for initial characterization. PT 81 specifically lysed 37% of Ficoll/Hypaque-prepared PBL depleted of monocytes by carbonyl iron ingestion. This antibody lysed 47% of T cells (PBL that formed dextran-enhanced, sheep erythrocyte rosettes; P less than 0.001) and 64% of a T cell subset (PBL that formed dextran-enhanced, porcine erythrocyte rosettes; P less than 0.001) when compared to cells treated with complement alone. Lysis of PBL with PT 81 plus complement caused a 100% enrichment in the number of surface immunoglobulin positive cells (P less than 0.01). The conclusion that PT 81 does not recognize B cells was further supported by double labelling experiments. Removal of PT 81 positive cells did not significantly affect the number of cells with C3b (zymosan-complement rosettes) or Fc gamma (erythrocyte-antibody rosettes) receptors. PBL that were treated with PT 81 plus complement were passed over Ficoll/Hypaque (density 1.09) to remove dead cells. The PT 81-depleted lymphocyte population displayed only 20%, 20%, 26% and 29% of the proliferative responses of control cells treated with complement alone (P less than 0.01) to several concentrations of the mitogens phytohemagglutinin, concanavalin A, soybean agglutinin and pokeweed mitogen, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)