C.-T. Chou

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.

Lymphokine-activated killer (LAK) cells: I. Age-dependent decline of LAK cell-mediated cytotoxicity.

Experiments were designed to assess age-related changes in generation of lymphokine-activated killer (LAK) cells and to test whether these changes can be modified by diets differing in the proportion of polyunsaturated to saturated fatty acids (P/S). Ficoll-Hypaque-isolated spleen lymphocytes of rodent chow-fed, 6-85-week-old C57BL/6 (H-2b), 8-81-week-old C57BL/10 (H-2b) and 6-62-week-old SJL (H-2s) mice were cultured in IL-2-containing medium and examined in 51Cr cytotoxicity assay. Similarly, Ficoll-Hypaque-isolated spleen lymphocytes of 6-36-week-old SJL mice fed diets which differed in the ratio of polyunsaturated/saturated fatty acids were cultured in IL-2-containing medium and assayed for cytotoxicity. Age-related decline of LAK cell-mediated cytolysis was observed in mice of both H-2b and H-2s haplotype. The age-related decline of LAK cell-mediated cytolysis was the consequence of age-related decrease in the rate of LAK cell precursor maturation. SJL mice fed from birth with diets differing in P/S did not differ in LAK cell-mediated cytolysis.

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.

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.

Volume, Adherence Properties, Membrane Antigens and Mitogen Responsiveness of Rabbit Lymphoid Cell Subpopulations

Subpopulations of rabbit spleen cells which respond to T and B mitogens, respectively, can be distinguished by sedimentation velocity in the earths gravitational field. T cell subpopulations which differed in their responsiveness to Con A and to PHA could be identified by differences in adherence properties and by their sensitivity to complement mediated cell kill with RTLA-antiserum.

Cross-reaction of a monoclonal antibody to human MHC class II molecules with rabbit B cells

Monoclonal antibodies, 21w4 and 44H10, against human MHC class II determinants, were analysed for their reactivity with rabbit lymphoid cells. Both MAb bind to all human B lymphoblastoid lines, irrespective of their HLA-DR phenotype. HLA-DR antigens, purified by affinity to 21W4 IgG Sepharose, can be precipitated with 44H10 MAb, indicating that both antibodies react with the same molecules. Competitive inhibition studies with purified MAb IgG show that 44H10 and 21w4 recognize different epitopes of HLA-DR molecules. The 21w4 MAb, previously shown to cross-react with cells of pig, mouse and sheep, does not react with rabbit lymphoid cells. The 44H10 MAb binds to lymphoid cell suspensions prepared from rabbit appendix, spleen and mesenteric lymph nodes. Its reactivity correlates with that of RABELA, a polyclonal antibody specific for rabbit B cells. Mesenteric lymph node and spleen cell suspensions, depleted of sIg+ cells, are devoid of 44H10+ cells, while similarly-treated appendix B cells still contain a subset of B cells which are sIg-, RABELA+ and 44H10+. These studies thus report on the presence of MHC class II determinants on rabbit B cells, cross-reacting with human HLA-DR.