S. Dubiski

A membrane antigen of rabbit thymus cells

Abstract Rabbit cells, bearing a thymus-specific antigen, which we call rabbit thymus lymphocyte antigen (RTLA), could be detected with a suitably absorbed heterologous antiserum (goat). In the presence of complement, the RTLA antiserum lysed more than 95% of thymus cells, 70 ± 6% of lymph node cells, 46 ± 10% of spleen cells and 12 ± 7% of bone marrow cells. The number of direct or indirect hemolytic spleen plaques was not reduced by treatment with RTLA antiserum and complement, but was greatly diminished by an unabsorbed thymus antiserum which killed more than 90% of bone marrow cells. RTLA-bearing subpopulations of spleen cells were characterized by velocity sedimentation analysis and were distinguished from Ig receptor bearing subpopulations. The antiserum concentration could be so adjusted that the cytotoxicity against bone marrow was not manifested, while the cytotoxicity against other cell populations remained unchanged. The latter were identified by thymidine incorporation induced by treatment with antibody directed against rabbit light chain allotype. A small subpopulation of thymus cells did not have RTLA antigen and sedimented with a velocity distinct from that of the peak of RTLA-bearing cells.

Mitogen Stimulation of Rabbit Spleen Cells before and after Complement-Mediated Cell Kill with an Antiserum Directed against the Thymus Antigen RTLA

Rabbit thymus lymphocyte antigen (RTLA) has been previously characterized as an antigen of rabbit thymus-derived cells (Cell. Immunol . 7: 484–501, 1973). It has now been shown that

Unequal expression of allelic allotypic specificities in circulating immunoglobulins, experimentally-elicited antibodies, and receptor-carrying cells

Abstract Unequal expression of allelic allotypic specificities “pecking order” (Ab locus) of heterozygotes has been attributed to an unequal number of cells bearing allotypically marked receptors. This hypothesis has been advanced to account for the allotype distribution in the indirect plaque-forming response of heterozygous animals immunized with sheep red cells and with dinitrophenol-conjugated proteins (Eur. J. Immunol.2, 391, 1972). In this paper various predictions, which follow from this hypothesis, have been tested. Immunization of A 4 A 5 rabbits with limiting quantities of antigen results in a high incidence of monoallotypic responders. The ratio of monoallotypic responders of A4 and of A5 type reflects the preponderance of A4 over A5 which is observed when animals are immunized with saturating doses of antigen. Animals of A 4 A 9 allotype, immunized with a single dose of antigen, and tested 8 days later usually made only A4 plaque-forming cells, and the incidence of monoallotypic A9 responders was as low as the percentage of A9 plaque-forming cells after immunization with two doses of antigen. The relative serum concentration of allotypically marked immunoglobulins also showed as marked “pecking order.” In general, it was as similar to the relative allotype proportion of plaque-forming cells which make antibody against sheep red cells. A possible exception to this general rule was the relative preponderance of A5 over A6. Also, the allotypically marked immunoglobulins of A 4 A 5 rabbits were more nearly equal in concentration than were their antibodies to red cells. Thus the “pecking order,” while not dependent on the identity of a particular immunizing antigen, might be modified by it. Direct tests were made to determine the number of cells bearing receptors of the two allotypic specificities of a heterozygote. Spleen cells from A 4 A 5 and A 4 A 9 animals were treated separately with antibodies directed against each of their Ab allotypic specificities and the consequent thymidine uptake was determined. On this basis, an allotype ratio of uptake could be evaluated. Ratios, obtained in this way, were similar to the allotype ratios of specifically elicited antibodies. It thus appeared that the unequal expression of allelic allotypic specificities in circulating immunoglobulins and experimentally elicited antibodies could be attributed to the higher number of cells carrying receptors of the predominant allotypic specificity.

A membrane antigen of rabbit bursal equivalent cells

Abstract Rabbit cells, bearing a specific antigen for bursal equivalent cells, could be detected with a suitably absorbed heterologous antiserum (goat). The antigen, detected with this antiserum, was designated RABELA. In the presence of complement, the RABELA antiserum lysed 80% of appendix cells, 50% of tonsil cells, 50% of spleen cells, and 25% of blood lymphocytes. It did not lyse a significant number of bone marrow or thymus cells. After complement-mediated kill with RABELA antiserum, cell populations lost the ability to respond to B mitogens. The responsiveness to the T mitogen, Concanavalin A, was reduced, but could be restored by addition of B cells which, alone, did not respond to Concanavalin A. RABELA-bearing subpopulations of spleen cells were characterized by velocity sedimentation analysis and were distinguished from subpopulations which took up thymidine after treatment with antibody directed against light chain allotypic specificity.

Age-related changes in interleukin production in BALB/cNNia and SJL/J mice and their modification after administration of foreign macromolecules

In vitro production of Interleukin-2, -3, -4 and -10 by activated lymphocytes of BALB/cNNia and SJL/J was studied. While IL-2 production in BALB/c mice remains constant throughout the life span of the animals (8-113 weeks), an increase in production from stimulated SJL cells was seen. Age-related increases in IL-3 and IL-4 production occur between young and middle age (8-60 weeks) in both strains. Some organ differences in quantity of lymphokine produced were seen; the direction of age-related changes was the same for lymphocytes of spleen and MLN. The exceptional feature of BALB/cNNia was the relative stabilization of the levels of interleukin production, as animals approach old age. BALB/cNNia and SJL, which are at the two opposite extremes of lifespan, differ also in their response to molecular interventions: in BALB/cNNia fetal sheep liver extract and hemocyanin increase the output of interleukins. This is in striking contrast to the effects observed in older SJL mice in which the extract reduced the output of IL-3 and IL-4 by old animals, whereas hemocyanin increased the output of IL-2 and IL-3 at all ages tested.

Purification of rabbit B spleen cells by removal of adherent and of T cells

B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against RABELA and RTLA, respectively. Other criteria depended on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.

Strain polymorphism in progression of aging: changes in CD4, CD8 bearing subpopulations.

Polymorphism of age-related changes in CD4 (L3T4) and CD8 (Lyt-2) determinants of spleen and thymus cells was assessed by fluorescence-activated flow cytometry. Cells from mice ranging from 5 weeks to greater than 2 years of age were examined. There is little age-related change in the proportion of CD4+ CD8- splenocytes in A, C57BL/6, DBA/1, DBA/2, and SJL mice (slopes 0.04, 0.06, 0.08, 0.17 and 0.17, respectively, when age in weeks was plotted against % of positive cells). Changes in the composition of the thymus are much more profound: CD4+ CD8+ cells of SJL mice decrease from 70% to less than 10% as the animals age from 5 to 69 weeks (slope -1.03), and in DBA/2 mice from 5 to 110 weeks (slope -0.88). While this decrease in CD4+ CD8+ cells occurs, there is a compensatory increase in CD4+ CD8- and CD4- CD8+ cells; this is a shift in the relative proportion of subpopulations rather than an increase in absolute cell numbers of a particular subpopulation. In contrast to the age-related changes of SJL and DBA/2 mice, there is relatively little change in the proportion of CD4+ CD8+ thymus cells in mice of strains C57BL/6, DBA/1 and A (slopes -0.03, -0.14 and -0.15, respectively).

A suppressor cell which interferes with antiallotype stimulated DNA synthesis of rabbit B cells

Abstract Responsiveness of rabbit spleen cells to anti-allotype antibody was measured in terms of increased thymidine incorporation. Incorporation was enhanced after removal of cells which had ingested or had adhered to magnetic particles. B lymphocytes, prepared from spleen cells by the removal of adherent cells and of RTLA bearing T cells, were more responsive to anti-allotype antibody than were the original spleen suspensions. This increase could not be explained by enrichment in B cells. It was concluded that an adherent cell suppressed B cell transformation. The addition of 2-mercaptoethanol to the cell cultures stimulated with mitogen augmented the incorporation of thymidine. Adherent cells interfered with 2-mercaptoethanol potentiation in the response to anti-allotype antibody but not in the response to Con A. Fractionation of spleen cells, over glass bead columns, yielded nonadherent and adherent cell populations. The responsiveness of nonadherent cells to anti-allotype induced thymidine incorporation was two to six times that of unfractionated cells. The responsiveness of nonadherent cells to stimulation by anti-allotype antibody was reduced after addition of adherent cells. Findings were discussed in terms of the inhibitory role played by adherent cells on anti-allotype antibody induced responsiveness of rabbit B cells and of the possible participation of a third cell type which functions as a promotor of mitogenic T cell stimulation.

Interactions between rabbit B and T lymphocytes in mitogenic response to staphylococcal protein A.

Abstract We have identified responder and helper cells involved in the response of rabbit lymphoid cells to stimulation with staphylococcal Protein A (SpA). Purified T but not B lymphocytes are activated by SpA. The response of T lymphocytes is enhanced by nonadherent B lymphocytes and by T lymphocytes. Lymphocytes irradiated or treated with mitomycin C retained at least one-half of their helper capacity. B lymphocytes contain cells which can only respond to SpA with the help of T lymphocytes.

BASILEA KAPPA LIGHT CHAINS OF RABBIT IMMUNOGLOBULINS

Immunogenetics of the Basilea allotype were investigated. It was shown that the Basilea gene is controlled at a locus identical with, or closely linked to, the Ab kappa allotypic locus. Basilea‐positive molecules have been physically separated by immunoabsorption from molecules carrying the λ chain markers, c7 and c21. Injection of newborn Basilea homozygotes with anti‐Basilea immune serum results in suppression of the synthesis of Basilea‐positive molecules, but does not affect the synthesis of the λ chain, or of the heavy chain allotypic markers. Allotype‐suppressed Basilea homozygotes do not recognize either the Basilea allotype or the kappa isotype as ‘self’ and make anti‐Basilea and anti‐kappa antibody upon immunization with Basilea antigen. The specificity of this antibody was identical with that of a heterologous anti‐kappa immune serum raised in a goat and made specific by absorption with purified lambda material. These results prove that Basilea gene products are of kappa isotype and are controlled at the Ab allotypic locus.