C57BL Cells Research Articles

Immunologgical self-tolerance in allophenic and embryo-aggregated mice

Allophenic mice, supposedly containing almost equal numbers of cells derived from embryos of mouse strains C57Bl and FVB, were shown in a recent paper to grow the B16 melanoma, a long transplanted tumor of C57Bl origin, much better than did mice of either the parental C57Bl strain or the C57Bl × FVB F1 hybrid. Mice containing smaller proportions of C57Bl cells rejected the tumor. A reconsideration of these suprising data, in light of the current literature, suggests that the better growth of the tumor in the 50-50% allophenics than in the C57Bl parental strain was almost certainly caused by the tumor stimulation engendered by a weak anti-C57Bl immune reaction in the overtly healthy allophenic mice.

Limited role of CD28-mediated signals in T helper subset differentiation.

The role of CD28-mediated signals in T helper cell maturation is not fully understood. We tested the requirement for costimulation through CD28 in several systems of CD4+, T cell differentiation. In vivo priming of mice with genetic disruption of CD28 (CD28-/-) yielded normal levels of antigen-specific interferon gamma production but markedly diminished levels of interleukin 4 (IL-4) after in vitro restimulation. In response to the pathogenic microbe, Leishman a major, C57BL6 CD28-/- mice were fully capable of controlling infection and exhibited a normal T helper 1 response. BALB/c CD28-/- mice unexpectedly exhibited normal susceptibility to L. major. BALB/c CD28-/- mice developed high levels of IL-4 mRNA and protein induction in the draining lymph nodes. In addition, susceptibility of BALB/c CD28-/- mice was reversed by neutralization of IL-4 in vivo. We also activated transgenic CD28-bearing T cells from the BALB and C57BL background in vitro in the presence of CTLA4Ig. BALB cells had greater IL-4 producing capacity than C57BL cells in the absence of costimulation. Diverse factors including costimulatory signals, genetic polymorphism, and the nature of the immunogen all influence T helper phenotype commitment, but these results provide evidence that CD28 is not an absolute requirement for generating either Th1 or Th2 responses.

Appearance of interfrontal bone in chimeric mouse.

The incidence of the interfrontal bone in mice differs between strains, being high in C57BL/6 and low in BALB/c. In the present study, aggregation chimeras (C57BL----BALB) were examined to reveal whether or not genetically different cells interact in the morphogenesis of the interfrontal bone. In C57BL/6 mice, large interfrontal bones appeared in almost all animals, whereas in BALB/c and reciprocal F1 crosses (C57 BL x BALB, BALB x C57BL), large bones were seldom observed while tiny bones at the inside of the skull appeared at a low incidence. In contrast, chimeras frequently had large interfrontal bones, the size of which varied considerably. Based on the degree of chimerism as determined by coat color mosaicism and glucose phosphate isomerase (G PI) analysis of tissues, the chimeric mice containing a dominant population of C57BL cells had large interfrontal bones, while those with a predominance of BALB cells had no bone or had tiny ones. The results indicated that the appearance of the interfrontal bone corresponded with the population of the C57BL cells occupying the skeletal rudiment, and that there was no interaction between C57BL cells and BALB cells in the morphogenesis of the interfrontal bone.

In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Spleen cells from male BALB/c mice infected 7 days earlier by an intraperitoneal injection of 3 X 10(4) PFU of a myocarditic strain of coxsackievirus B-3 lysed virus-infected endothelial cells in a 51Cr release assay. Cytotoxic activity in the in vivo sensitized spleen cell population could be further increased by culturing the immune spleen cells from infected mice on virus-infected or uninfected endothelial cells for 6 to 7 days in vitro. Cytotoxicity of in vitro cultured spleen cells to infected targets was mediated by T lymphocytes since reactivity was abolished by treatment of the spleen cells with anti-thy 1.2 serum and complement. Reciprocal assays with BALB/c and C57BL cells indicated that maximum cytotoxicity occurred when spleen cells were sensitized on syngeneic endothelial cells. Other experiments showed that spleen cells sensitized to coxsackievirus B-3 or encephalomyocarditis virus were selectively cytolytic to targets infected with the homologous virus. Adoptive transfer of T cells cultured in vitro on infected endothelial cells retained their ability to induce myocarditis in T-lymphocyte-deficient mice.

The effect of T lymphocyte depletion on neonatal tolerance induction, graft-vs.-host disease and cellular chimerism.

Treatment with a monoclonal anti-Thy-1 antibody and complement completely prevented C57BL spleen cells from causing graft-vs.-host disease following their inoculation into newborn CBA mice. The proportion of mice that became tolerant to C57BL antigens, as measured by skin grafting, was significantly less compared with mice given (CBA X C57BL)F1 hybrid cells. This was not due to the elimination of T cells, for antibody-treated F1 cells induced tolerance as readily as complement-treated control F1 cells. To investigate whether the apparent superiority of F1 cells over C57BL cells is attributable to differences in the mechanism inducing and maintaining unresponsiveness, two approaches were followed. First, the level of donor cell chimerism in the spleens of tolerant animals was studied. Though no difference between F1 and C57BL cells was uncovered, the presence of T cells in the donor inocula favored the establishment of chimerism. Second, the involvement of suppressor T cells was examined in adoptive transfer experiments. Splenic suppressor T cells were associated with tolerance regardless of how it was elicited. Preliminary results with F1 cells show that the tolerogenic property is not confined to any one cell type. It is proposed that the greater tolerogenicity of F1 cells is brought about by the presence of host-type (self) antigens, which enable the tolerogenic signals to operate without recourse to antigen processing by host cells.

Tolerance, immunocompetence, and secondary disease in fully allogeneic radiation chimeras.

The aim of this study was to ascertain the extent to which secondary disease and mortality in fully allogeneic chimeras (C57BL leads to CBA) is caused (if at all) by a delayed graft-versus-host reaction. Adult CBA males were thymectomized, irradiated, and reconstituted with T-lymphocyte-depleted C57BL or CBA bone marrow cells (BMC), followed three weeks after irradiation by implantation under the kidney capsule of thymic lobes from C57BL or CBA fetal or adult donors. These mice were observed for the development of secondary disease for periods in excess of 250 days, and they were examined at 5 weeks or 4 months for T lymphocyte reactivity and tolerance to alloantigens, using the cell-mediated lympholysis assay (CML). The following results were obtained. First, removal of T lymphocytes with anti-Thy 1 antibody and complement from allogeneic bone marrow did not prevent wasting and eventual death, although it prolonged the lifespan of mice substantially. Second, T lymphocytes generated from bone marrow-derived precursor cells became tolerant of the histocompatibility antigens of the thymus donor strain but remained normally reactive to third-party antigens. Third, allogeneic radiation chimeras did not survive as well as animals reconstituted with syngeneic cells, even when they were demonstrably tolerant in CML. Fourth, C57BL BMC maturing in a CBA host equipped with a C57BL thymus graft did not become tolerant of host antigens, indicating that extra-thymic tolerance does not occur in fully allogeneic--as opposed to semiallogeneic--chimeras. It is argued that the function of B lymphocytes and/or accessory cells is impaired in fully allogeneic radiation chimeras, and that the mortality observed was directly related to the resulting immunodeficiency. The relevance of the results described in this paper to clinical bone marrow transplantation is discussed.

Replication of spleen focus-forming friend virus in fibroblasts from C57BL mice that are genetically resistant to spleen focus formation

Induction of erythroleukemia and spleen focus formation by the defective spleen focus-forming component of Friend virus (SFFV) is governed by a genetic locus of the mouse termed Fv-2 ( F. Lilly, J. Nat. Cancer Inst. 45, 163–169, 1970 ). Subsequently, the Fv-2 gene was reported to act by directly controlling SFFV, but not helper virus replication in the animal ( R. A. Steeves, R. J. Eckner, M. Bennett, E. A. Mirand, and P. J. Trudel, J. Nat. Cancer Inst. 44, 1209–1217, 1971 ; R. A. Steeves, F. Lilly, G. Steinheider, and K. Blank, In “Differentiation of Normal and Neoplastic Hemopoietic Cells” (B. Clarkson, P. A. Marks, and J. Tills, eds.), pp. 591–600. Cold Spring Harbor Conference on Cell Proliferation, Cold Spring Harbor, N. Y., 1978 ) or in cultured cells ( N. M. Teich and T. M. Dexter, In “Oncogenic Viruses and Host Cell Genes” (Y. Ikawa and T. Odaka, eds.), pp. 263–276. Academic Press, New York, 1979 ). To determine how Fv-2 controls SFFV-induced erythroleukemia, we have examined its effect on SFFV replication in fibroblasts of resistant C57B1 mice infected with an ecotropic SFFV-Friend leukemia virus pseudotype. It was found that virus produced by C57B1 cells had spleen focus-forming activity in susceptible mice. RNA isolated from virus produced by C57Bl cells was indistinguishable in electrophoretic mobility and RNase T,-resistant oligonucleotides from authentic SFFV RNA described previously. We conclude that the Fv-2 gene does not inhibit SFFV replication in fibroblasts of Fv-2-resistant C57BI mice. Based on previous work of others and on our results, we suggest that Fv-2 acts either by controlling in hematopoietic cells susceptibility to helper virus, or by controlling a target necessary for transformation. Loss of SFFV from SFFV-helper virus stocks passaged in resistant animals would be a consequence of selection against, rather than direct inhibition of, SFFV. In susceptible animals, rapidly proliferating, SFFV-transformed cells would maintain a high ratio of SFFV to helper virus. Lack of transformed cells in resistant animals, and the consequent lack of selection for SFFV, would favor helper virus over SFFV.

Antibody formation against antigen-recognizing receptors of T lymphocytes in a syngeneic system

Antibody formation against antigen-recognizing receptors of T lymphocytes were shown to be capable of being formed in a syngeneic system. Antiserum of CBA mice receiving intravenous injections of CBA lymphocytes immune against C57BL cells specifically inhibited blast transformation of CBA T lymphocytes against C57BL cells only in a mixed culture. The same antiserum had no effect on proliferative actiivy of CBA T lymphocytes reacting to “foreign antigen” — i.e., DBA/2 cells. No antibodies against C57BL cells likewise were found in the antireceptor antiserum. A regulatory influence of autoantireceptor antibodies on the immune response is postulated.

Influence of an M-locus difference on the cellular and humoral immunological reactivity against H-2 determined antigens of the mouse.

Studies were initiated to determine whether an immune response to the Mls antigen of C3H mice could modify responses of CBA lymphocytes (H-2-compatible) to a foreign H-2 complex. CBA lymphocytes, partially tolerant to the C3H-determined Mls antigen, generated less effector cell activity against C57Bl cells (H-2-incompatible) than lymph node cells from normal CBA donors when infused into irradiated C3H X C57Bl hosts. Effector cell activity was measured as the capacity of the cells in the irradiated spleens to inhibit CBA X C57Bl bone marrow proliferation. In contrast, immunization of CBA mice with C3H X C57Bl cells yielded lower antibody titers against C57Bl cells than immunization with CBA X C57Bl cells. Furthermore, preinjection of CBA mice with C3H X CBA cells strongly reduced the capacity of the animals to produce antibodies against C57Bl cells. Thus, these data support the conclusion that an immune response to a foreign Mls antigen may either enhance or suppress an immune response to H-2-incompatible cells.

Partial tolerant state against H-2 disparate cells. No impaired specific reactivity in MLC, GVH, or antibody production.

Injection of CBA mice with H-2-compatible lymphoid cells from C3H hybrids induces a specific reduction of the mixed lymphocyte culture (MLC) response of their lymphoytes. This is not the case after injection of H-2-disparate C3H-hybrid cells, presumably because they are rapidly eliminated due to the immune response of the host. This investigation shows that CBA mice injected with CBA X C57Bl cells (H-2-disparate) at an age of 0-3 days, but not older, develop a specifically reduced MLC response after infusion of C3H X C57Bl cells as adults, indicating that they were tolerant to the C57Bl-determined antigens. However, lymphocytes from such mice showed a normal reactivity against C57Bl as assessed by MLC, graft-versus-host tests, and capacity to produce specific antibodies.

Complex nature of the production of 'effector' cells and their interaction with target cells in the M-antigen system of the mouse.

The capacity of lymphocytes from mouse strain CBA to generate 'effector' cells against the H-2-compatible, M-antigen-incompatible strain C3H and their interaction with such target cells were investigated. It was observed that CBA lymphocytes injected in, and 5 days later obtained from, the spleens of irradiated C3H X CBA hybrids ('educated cells') could strongly inhibit the growth of C3H X CBA bone marrow cells but were almost nonresponsive to C3H X C57BI bone marrow targets (H-2-incompatible). CBA lymphocytes educated in irradiated C3H X C57BI hosts displayed reactivity against C3H X C57BI and CBA X C57BL but not against C3H X CBA bone marrow target cells. Additional tests indicated that the M antigen determined by C3H is expressed to approximately the same extent on C3H X CBA and C3H X C75BL cells and that the M antigen on C3H does not cross-react immunologically with antigens on C57BL cells. Moreover, it was observed that CBA X C57BI lymphocytes were triggered to cell proliferation by C3H antigens but were unable or had a highly reduced capacity to develop 'effector' cells in response to this antigenic stimulus. These results indicate that generation of 'effector' cells and their interaction with target cells are very complex processes.

Formation of plaques by adherent spleen cells from nonimmunized mice, incubated in vitro with the nonadherent fraction of immune allogeneic cells.

Twenty-four-hour incubation of adherent spleen cells from normal C57BL mice with purified nonadherent spleen cells from CBA mice, in the peak of their primary response against sheep red blood cells, resulted in the appearance of plaque-forming cells in the adherent cell population. The immunological nature of these plaques was demonstrated by their complement dependency and the inhibitory effect of specific anti-mouse IgM serum. Direct, but not indirect plaque formation was found to be associated with the adherent cell population for which direct cell to cell contact is necessary. Experiments with cytotoxic specific anti-strain sera revealed that in this system, the adherent plaque-forming cells are the nonimmune adherent C57BL cells.

Participation of three cell types in the anti-sheep red blood cell response in vitro. Separation of antigen-reactive cells from the precursors of antibody-forming cells.

Spleen cells of unprimed CBA mice were shown to produce anti-sheep red blood cell antibodies comparable in amount in vivo and in vitro. Under identical culture conditions spleen cells of C57BL mice did not respond. CBA spleen cells, passed through columns of cotton wool (CBAf), were equally inactive in vitro. However combined cultures containing both CBAf and C57BL cells yielded as many or more plaque-forming cells than the same number of unfractionated CBA spleen cells. Analysis of the contribution of each cell population to the synthesis of antibody in the combined cultures has disclosed the participation of three cell types. A thymus-dependent, radiosensitive cell was derived from the CBAf population, while the C57BL was the source of the precursor of the antibody-forming cell and of a radioresistant cell. The latter two were partially separated in a Staput apparatus.

An experimental system for the simultaneous estimation of mitostatic and lymphotoxic effects of immunosuppressants and cytostatics.

Sublethally (600 R) irradiated (CBA x C57BL)F(1) mice were grafted intravenously with parental lymph node cells in doses ranging from 0.2 x 10(6) to 12 x 10(6). The transplantation of these lymphoid cells leads to inactivation of the recipient's endogenous CFU (as measured by the diminution of the number of colonies registered on the 10th day after irradiation). A 50% inactivation was observed when the graft size of the CBA cells was 0.52 x 10(6). This figure for C57BL cells was 10 times more. This experimental system evaluates two simultaneously developing processes: the multiplication of endogenous CFU and the homograft reaction of transplanted lymphocytes against them. Both processes can be quantitatively estimated simultaneously in the same experiment by the determination of the number of colonies in corresponding experimental groups. Thus it was possible in a single experiment to compare quantitatively the effect of immunosuppressants on two points: (a) mitostatic action (suppression of CFU) and (b) lymphotoxic action. The latter, a true immunosuppressive effect, represents suppression of GVH activity of lymphoid cells and is demonstrated by abolition of the inhibition of endogenous colony formation. In the present system we have tested 6-MP, ALS, cyclophosphamide, hydrocortisone, and other drugs. The definite mitostatic and lymphotoxic doses of drugs are ascertained. Cyclophosphamide and ALS proved to be drugs with high dose ranges of selective lymphotoxic action. Hydrocortisone acetate had a more narrow range of selective lymphotoxic effect. 6-MP and Imuran (azathioprine) failed to exert any selective action on lymphoid elements. They possessed pronounced mitostatic efficiency, however.

Interstrain somatic cell hybrids in the mouse: Chromosome and enzyme analyses

An intraspecific mouse somatic cell hybrid population (C3H X C57BL) and its clonally derived descendants were studied. The modal chromosome number of the cloned hybrid decreased by 12% after 86 tissue culture passages, and by 20% after 2 years of tumor passage. This moderate loss of chromosomes represented a genuine reduction and could not be attributed to the centric fusion of acrocentric chromosomes. Intraspecific isoenzyme genetic variants specific for and different between C3H and C57BL cells were retained in the hybrids. The enzyme variants could be employed to deduce the balance between the input parental genomes in the hybrids.

Modification of an incompatible graft-versus-host relationship by admixture of grafts with F1 haemopoietic cells.

SummaryWhen lethally-irradiated CBA.T6T6 mice were grafted with mixtures of C57BL and (C57BL × CBA.T6T6)F1 cells of bone-marrow or foetal liver the incidence of secondary disease was considerably reduced as compared with mice receiving the incompatible C57BL cells only. Cytogenetic examination of bone-marrow, spleen, thymus and lymph node and identification of CFU in blood showed that F1 cells were not eliminated as anticipated and that C57BL cells persisted in similar numbers, whether or not the host had secondary disease. The mechanism of avoidance of secondary disease in these experiments appears to be adaptation of the incompatible allogeneic graft, the host being carried through the periods of haemopoietic and immunological insufficiency by the F1 cells. The findings constitute not only a therapeutic advance in a hitherto incompatible graft-versus-host relationship but also require reappraisal of the dominant role accorded to GVHR in secondary disease.

RELATIVE ABILITY OF PARENTAL MARROWS TO REPOPULATE LETHALLY IRRADIATED F1 HYBRIDS.

The relative abilities of C57BL and 101 marrow to repopulate the hematopoietic system of lethally x-irradiated mice was determined. The disproportionately greater ability of 101 than C57BL cells to repopulate (C57BL X 101) F/sub 1/ recipients is reported, and data are given on the relative ability of marrow from other strains of mice to repopulate lethally irradiated mice. Consistent with the observation that C57BL marrow is less efficient that 101 marrow is the finding that the number of C57BL cells required to produce functioral long-terra marrow grafts was much larger than the number of 101 cells. Factors that might influence the differential in the growth ability of the two types of marrow are discussed. (H.M.G.)

The immunological response produced in inbred mice following the isologous and homologous transfer of antigenically stimulated spleen cells.

SUMMARYAntigenically stimulated spleen cells from C3H and C57Bl mice have been transferred to isologous and homologous recipients and both the immediate appearance of antibody and the response to a challenge injection of antigen 11–12 weeks later have been studied.Antibody can be detected as early as two days after isologous transfer of C57Bl cells, but none appears after homologous transfer. Isologous transfer of C3H cells gives much less evidence of early response showing either a delay up to six days before first appearance or failing completely.Following late challenge mice that received isologous cells responded with typical secondary response in the great majority. No secondary type responses were obtained with homologous combinations.

Immunogenetic Studies on X-Irradiated Mice Treated With Hematopoietic Cells and Grafted With Tumor Tissues<xref ref-type="fn" rid="FN1">2</xref>

A study was made of the behavior of tumor grafts in the radiation chimera to analyze the immune reactivity of the donor's hematopoietlc cells and the influence on tumor cells of the radiation chimera. The regression of homografts of Sarcoma C10 and Sarcoma MC1M (of C3H origin) in irradiated C57BJ animals treated with C57BL cells and their progressive growth in irradiated animals treated with C57BE fetal hematopoietic cells gave evidence of the ability of donor cells to form transplantation immunity against foreign antigens, thus substantiating the graft versus host theory as the basis for homologous secondary disease. Tumor grafts produced metastases in irradiated animals, and irradiation and immunogenetic factors were shown to influence their development. The transplantation of Sarcoma SBL1 into various types of genetic chimeras was accompanied by immunogenetic changes in the tumor, which were manifested by the loss of strain specificity. The effect of total-body irradiation of the host and the absence of isologous C57BJ cells were shown to be the main factors determining this change. Their analysis provided definite indications that the acquired homotransplantability of SBLx sublines is basically a result of immunogenetic transformation induced by the host and not a result of selection of pre-existing compatiblemore » cell variants. (auth)« less

Immunogenetic Studies on X-Irradiated Mice Treated with Homologous Hematopoietic Cells<xref ref-type="fn" rid="FN1">2</xref>

ing place in x-irradiated mice treated with homologous hematopoietic cells. The results of experiments with hybrid combinations of hosts and donors suggest that the grafted cells react imnnunologically against the isoantigens of the irradiated recipient. The production of antiC3H agglutinins by the inoculated C57BL cells was demonstrated by the transfer of lymph nodes from the irradiated and treated C3H mice back to normal C57BL mice. The ''cytotoxic'' anti-host immune reaction responsible for the lethal ''secondary disease'' of homologously treated mice was demonstrated by the passive transfer of tramsplantation immunity produced by the isoantigens of the recipient. Bone marrow from irradiated C3H mice treated with C57BL cells conferred on other irradiated C3H mice a much shorter survival time than normal C57BL cells. It is concluded that the cells from the C57BL-treated C3H donors were immunologically activated by the primary C3H hosts and killed the secondary C3H hosts through an immunologically secondary response. Thc mortality of nvice treated with mixed embryonic isologous and adult homologous cells indicates that even if, theoretically, a delayed antigraft response is feasible, the graft antihost response has a domin:int role in the immunogenetic incompatibility. An attempt to overcome this incompatibility was made by injecting irradiated C3H micemore » with fetal C57BL liver cells. The increased survival time obtained in this experiment may suggest a method for the application of the principles of biological protection to animals of genetically heterogeneous populations. (auth)« less