Induction and abrogation of unresponsiveness in nude mouse cells.

Nude mice were injected with antigen and T cells at different times to induce unresponsiveness to SRBC. Spleen cells derived from these mice were tested in vitro for the capability to produce antibody-forming cells against sheep erythrocytes in the presence of a T-cell-replacing factor. It was found that priming with antigen alone did not result in paralysis but a later injection of thymus-derived lymphocytes together with antigen results in unresponsiveness of these cells in vitro, provided there was an interval of several days between the in vivo administration of thymus lymphocytes and the explantations of cells to in vitro cultures.

The transplantation barrier of nude mice.

Syngeneic memory cells can be stimulated to yield a secondary immune response after their transfer into irradiated euthymic recipients as well as into young thymusless nude mice. It is shown that nude mice older than twelve weeks of age are not permissive towards memory cell activation as it is found in non-irradiated euthymic animals. This barrier to isogeneic or congeneic cells seems to be caused by a pool of cyclophosphamide-sensitive cells. Since young nude mice could be rendered as unpermissive as older nude mice by pretreatment with either PNA-agglutinable thymus cells or nylon-wool passed spleen cells, it is suggested that an increased number of precursor T cells in older nude mice might induce this effect. Further experiments with monoclonal antibodies against the Lyt-1, Lyt-2, and L3T4 marker on T cells indicate that T-helper/inducer activity might be required to establish the "isogeneic barrier" in nude mice.

Membrane-associated nucleotide pyrophosphatase activity on leucocytes from different compartments.

The distribution of membrane-associated nucleotide pyrophosphatase activity has been investigated on mouse leucocytes of different origin. It is shown that enzyme-specific activity is higher on bone-marrow cells than on cells derived from peripheral organs. Since thymus lymphocytes show the lowest activity, it is obvious that the thymus environment can influence the enzyme activity on bone-marrow stem cells which have entered this organ. Spleen cells from thymus-deficient nude mice displayed more enzyme activity than spleen cells from normal mice, although the percentage of lymphocytes was similar in both donor-cell populations.

Functional maturation of neonatal spleen cells.

Maturation of neonatal spleen cells was studied in vitro with a cell population restricted with respect to functional properties. It is shown that the onset of the immune response to SRBc in post-natal mice was delayed because B and T cells were incompetent. It appears, however, that the development of these two cell populations does not occur in parallel. Since the addition of adult macrophages failed to overcome the incompetence of neonatal B cells in the presence of a T cell replacing factor, it is suggested that the late appearance of immune competence is due to the inability of B cells to process a T-cell signal.

Evidence for a mutual influence of idiotype-expressing donor cells and recipient lymphocytes in neonatal hosts.

Nonirradiated recipients do not permit activation of transferred syngeneic lymphocytes. This transplantation barrier gives us insight into the mechanism of the regulatory circuits of the donor's and host's immune network. The present report demonstrates that this barrier depends on the state of differentiation of cells from both donor and host. We measured the expression of anti-alpha (1-3)dextran (Dex) correlated idiotypes by cells from responder BALB/c animals (allotype IgHa, Dex+, IdDex+) in nonresponder (IgHb, Dex-, IdDex-) congeneic mice. Adult IgHb recipients did not permit activation of either adult spleen or bone marrow cells (isogeneic barrier). Neonatal recipients showed functional tolerance towards adult spleen cells. By contrast, neonates neither permitted activation of adult B cell-depleted bone marrow cells, nor of neonatal immature spleen cells. The results show that the immature system of the neonate is permissive towards mature (adult) congeneic lymphocytes, but not towards immature cells from congeneic neonates or adult bone marrow. It appears that mature adult cell populations are dominant in the immature neonatal host. However, the time required by transferred immature cells to differentiate in the neonatal recipient concomitantly allow the latter to gain maturity and functionally reject the grafted cells.

xid mice fail to express an anti-dextran immune response but carry alpha(1-3)dextran-specific lymphocytes in their potential repertoire.

Even after repeated immunizations with alpha(1-3)dextran (Dex) male (CBA/N x BALB/c)F1 mice fail to produce specific antibodies whereas nondefective female littermates express an idiotype-positive anti-Dex immune response. This failure of xid mice to express serum anti-Dex immunoglobulins is not only limited to immunization with the thymus-independent (TI-2) antigen Dex, since immunization with anti-idiotypic antibodies against Dex-specific idiotypes does not overcome this defect. When xid lymphocytes are cultured in the presence of mitogens, in vitro anti-Dex responses are markedly reduced. We show here that alpha(1-3)Dex-specific hybridomas can be established by fusion of splenic lymphocytes from xid mice to Sp2/0 myeloma cells. Therefore, these mice do carry the potential to generate B cells specific for Dex. All hybridoma antibodies were found to be of IgM isotype, bearing the lambda light chain typical for alpha(1-3)Dex-specific antibodies. Whereas monoclonal anti-Dex antibodies obtained from spleen cell hybridomas from female littermates showed variable idiotope patterns, hybridoma proteins from the immune-defective NBF1-xid mouse expressed only a limited pattern of Dex-specific idiotopes, suggesting that these hybridomas derived from a common precursor.

Non-specific T-cell replacing factor fails to promote a secondary response in vivo.

The supernatant from concanavalin-A-treated cultures has been used in vivo in nude mice to investigate the nature of the antibody response that can be induced after injection of this non-specific T-cell replacing factor (NSF). Although NSF alone does not induce a response, it permits a greatly augmented response to sheep erythrocytes. This response is, however, restricted to IgM and is not accompanied by development of memory. No secondary response could be obtained with antigen alone or with NSF and antigen, and there was usually no response even if normal congenic thymus cells were injected with antigen into animals that had responded to NSF and antigen.

In vivo activity of a nonspecific T cell-replacing factor.

Nonspecific T cell-replacing factors prepared as supernatants from mixed lymphocyte cultures or concanavalin A-stimulated spleen cells are active in vivo iv injected into nude mice at least 3 days before antigen. The supernatants appear to act by enhancing the week IgM responses that occur in untreated nudes. Secondary responses and IgG antibody were not found.

B cell dependence of the congeneic barrier towards idiotype-carrying cells.

Immune cells transferred into nonirradiated animals of the same genotype face a barrier which severely affects their capacity to function in the host. We studied this phenomenon in allotype-congeneic animals. When anti-dextran immune cells of the responder strain (BALB/c-Igha) are injected into an allotype-congeneic host (BALB-Ighb) the grafted cells are suppressed to give an idiotype-positive response. This congeneic barrier of thymus-independent immune cells is lost in mice carrying an X-linked B cell defect: when idiotype-positive immune cells from responder (CBA/N X BALB/c)F1 mice are transplanted into congeneic nonresponder animals (CBA/N X BALB-Ighb)F1, female recipients are nonpermissive towards grafted cells whereas B cell-defective male littermates allow donor cells to develop an idiotype-positive anti-dextran response. These results show that the congeneic barrier towards dextran-immune cells is related to the maturation stage of B cells in the recipient. Since B cell-defective (CBA/N X BALB/c)F1 animals do not display an anti-idiotypic response in contrast to intact littermates and because CB16-KN mice are nonpermissive towards CB8-K lymphocytes, differing in VH genes only, we suggest that the "isogeneic barrier" depends on a mechanism recognizing VH structures.

Demonstration of glutamate dehydrogenase isozymes in beef heart mitochondria.

Glutamate dehydrogenase (GDH) has been purified from beef heart mitochondria and compared with crystalline beef liver GDH. The specific activity of heart GDH was 127 units and of liver GDH 80 units. Heart GDH subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis had a protein corresponding to liver GDH and a smaller molecular weight protein. On agarose gel electrophoresis heart GDH activity was resolved into two fractions (with or without protease inhibitors) while liver had only one fraction. One of the heart fractions moved with liver GDH on electrophoresis. Thermal stability studies showed heart and liver GDH activity differed. Mouse antibodies to liver GDH precipitated both liver and heart GDH on double immunodiffusion. Mouse antibodies to liver GDH identified on nitrocellulose paper the polypeptide band of liver and heart GDH that were the same molecular weight but did not cross-react with the smaller molecular weight polypeptide present in heart GDH. Trypsin digestion of the two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GDH from beef heart mitochondria did not show any overlapping peptides. We conclude beef heart GDH activity is composed of two isozymes. One is the same as beef liver GDH, and the other is a smaller molecular weight protein. We propose the terms GDH-LM for the liver GDH isozyme and GDH-HM for the smaller molecular weight isozyme present in heart mitochondria but not liver.