Cellular aspects of tolerance. VI. The effect of age on responsiveness and tolerance induceability of SJL mice

New born and 3-week-old SJL mice but not 8–12-week-old animals could be rendered tolerant to rabbit γ-globulin. Animals reconstituted with thymus cells from 12-week-old donors and bone marrow cells from 3-week-old donors showed resistance to tolerance induction. Animals reconstituted with bone marrow cells from 12-week-old animals and thymus cells from 3-week-old donors could be rendered tolerant. Earlier work has shown that tolerance could be induced in older animals, if they were deprived of competent accessory cells. It was suggested that a lesion in the thymus cell population is expressed through the mediation of accessory cells. The possibility of a relation between resistance to tolerance induction and lymphoid malignancies was discussed.     

Cellular events in tolerance. II. Thymus-bone marrow cell cooperation in the immune response to BSA in Wistar Furth rats

Normal bone marrow cells from Wistar Furth rats were competent to transfer immune responsiveness to bovine serum albumin to thymectomised, irradiated, syngeneic recipients. When the bone marrow cells were taken from donors thymectomised early in life they were incompetent, but competence was restored by addition of normal thymus cells. It was concluded that normal Wistar Furth bone marrow cells contain some thymus-derived cells. Thymus cells from tolerant donors were less effective in cooperation with bone marrow cells, however the thymus cells appeared less tolerant than their donors.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

Immunocompetent cells in the chicken. II. Synergism between thymus cells and either bursa or bone marrow cells in the humoral immune response to sheep erythrocytes

Newly hatched F₁ hybrid chicks isogenic for the strong B histocompatibility locus were rendered immunologically incompetent by cyclophosphamide treatment and x-irradiation. They were then injected intravenously with thymus, bone marrow, or bursa cells together with sheep erythrocytes (SE) and received another iv injection of SE 3 days later. Splenic plaque-forming cells (PFC) and serum hemagglutinins were assayed 7 days after transfer. At donor ages of 14–26 days, cells from thymus (T) and bone marrow (BM) showed synergism when injected together, as indicated by a significantly higher geometric mean of PFC per recipient spleen in the BM + T group than in the BM group. The response of the T group was extremely low. With thymus and bursa cells from 6- to 28-day-old donors, significant synergism was demonstrated in 3 of 9 individual experiments. However, almost all the other 6 experiments showed marked differences in the same direction, and the combined probability for all experiments was < 0.001. The most striking demonstration of thymus + bursa synergism was made in 2 experiments using 1-week-old donors. Bone marrow cells from 1-week-old donors failed to cooperate with thymus, as did BM cells from older bursectomized agammaglobulinemic donors. This suggests that B cells from bone marrow originate in the bursa. Thymus-bursa cooperation was somewhat difficult to demonstrate in individual experiments using donors over 1 week of age, owing to the occurrence of some responses with bursal cells alone and to variability of response within bursa or bursa + thymus recipient groups. Synergism between thymus and bursa cells was more consistently demonstrable when irradiated normal spleen or low doses of bone marrow cells were added. These additions led to an increased response and a lowered coefficient of variation in the thymus + bursa recipient groups. The ‘third’ cell type needed for optimal response by the thymus and bursa cells together was tentatively identified as a macrophage.     

A bicellular mechanism in the immune response to chemically defined antigens. 3. Interaction of thymus and bone marrow-derived cells

The role of thymus and bone marrow-derived cells in the in vitro response to the dinitrophenyl (DNP) determinant was studied using the millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by the technique of inactivation of DNP-coupled T-4 bacteriophage. It was found that spleens of mice total-body irradiated at 750 R, treated with bone marrow and thymus cells after exposure and immunized against rabbit serum albumin (RSA) were able to produce antibodies to DNP when challenged in vitro with DNP-RSA. Such a response was not produced by spleen explants from x-irradiated mice treated with either thymus or bone marrow cells. Neither were antibodies to DNP produced by spleens of animals repopulated with thymus and bone marrow cells, but not immunized with the carrier. This carrier effect was manifested when the irradiated mice were treated with RSA and thymus cells 6–8 days before administration of the bone marrow cells. Yet, such an effect was not observed when the RSA and bone marrow cells were given 6–8 days before injection of the thymus cells. Thus, the thymus-derived cells appear to play the role of cells sensitive to the carrier (RSA), whereas the bone marrow seems to be involved in the production of antibodies.     

Cell-Cooperation in Anti-Sheep Red Blood Cell Antibody Response in Mouse Spleen Cell Cultures

Restoration of impaired antibody response to sheep red blood cells (SRBC) in spleen cell cultures from mice treated with heterologous antilymphocyte globulin (ALG) was studied by adding normal cells from various sources, to explore the problems of cell-cooperation in anti-SRBC antibody response and the target of ALG. When spleen cells from ALG-treated mice were separated into macrophage-rich and lymphoid cell-rich subpopulations, only the latter was found to be impaired in the ability for anti-SRBC antibody response. Addition of even a small number of normal allogeneic spleen cells sufficiently restored the impaired anti-SRBC antibody response of the spleen cells from ALG-treated mice. By use of allo-antisera, most hemolysin plaque-forming cells (PFC) generated in such cultures were proved to be derived from the cells of ALG-treated mice. Restoration was also achieved by adding thymus-derived cells, which were obtained from spleens of mice heavily irradiated and repopulated with syngeneic thymus cells, or lymphoid cells directly collected from thymuses. All results indicate that ALG selectively depletes the thymus-derived antigen reactive cells (ARC) in the spleen cell population, and that ARC supplied from normal spleen or thymus can interact with plaque-forming cell precursors (PFCP) that remain intact in the spleen cell population of ALG-treated mice. The results also suggest that a single ARC interacts with more than one PFCP and makes them develop into PFC.     

Cellular aspects of tolerance. III. The responsiveness of T cells from tolerant donors after exposure to a cross-reacting antigen

Mice, rendered tolerant to rabbit gamma globulin (RGG), were immunized with RGG or with dinitrophenylated RGG (DNP₄₀-RGG), incorporated in adjuvant. The resulting response was evaluated in terms of the half-life of trace labeled RGG (¹³¹I-RGG). An antibody response against the tolerance inducing macromolecule could be elicited with DNP₄₀-RGG, but not with RGG. Reconstitution experiments revealed that thymus derived (T) cells from tolerant donors could cooperate with bone marrow cells from normal donors in the response elicited by DNP₄₀-RGG, but could not effectively cooperate with bone marrow derived (B) cells from tolerant donors. Such B cells could cooperate with T cells from normal donors. The relative difference between native and chemically modified proteins played an important role in this tolerance circumvention, since analogous experiments with human instead of rabbit gamma globulin did not result in an effective response to determinants of the tolerance-inducing proteins. It was suggested that the number of effectively immunogenic determinants on DNP₄₀-RGG was low in B and in T cells of animals tolerant to RGG and that the probability of effective cooperation was consequently extremely low. If one of the two cell types came from a normal animal and thus could respond to a large number of determinants, the probability of effective cooperation increased so as to reveal the responsiveness of the “tolerant” cell population. There was no indication that the responsiveness of the tolerant T cell population was directed against tolerance-inducing determinants.     

Cellular aspects of tolerance. I. Parameters of tolerance induction in T cells of spleen and thymus

Unresponsiveness of T cells in thymus and spleen of tolerant animals was determined by reconstitution of lethally irradiated recipients. The degree of responsiveness of these animals was assessed by antigen elimination and two types of plaque assays (liquid and agar) with different sensitivity. Unresponsiveness occurred more rapidly in T spleen cells than in thymus cells. Unresponsiveness of T cells could be induced in the spleens of thymectomized animals and in T cell repopulated thymectomized lethally irradiated recipients. Induction of unresponsiveness did not depend on proliferating bone marrow cells or on accessory cells.     

Antibody formation in mouse bone marrow. II. Evidence for a memory-dependent phenomenon

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity in a primary response to sheep red blood cells (SRBC), while PFC activity in the secondary response to SRBC can be clearly demonstrated. This phenomenon was studied by means of cell transfer experiments.T cells, which are involved in an anti-SRBC PFC response, were shown to be very scarce in normal mouse bone marrow. This is considered to be the cause of the low PFC activity in the marrow during the primary response to SRBC.In normal mouse bone marrow precursors of IgM-PFC but not of IgG- and IgA-PFC could be found. Priming with SRBC induced the appearance of IgM-, IgG-, IgA- and T-memory cells in the marrow. These B- and T-memory cells were shown to be specific for the antigen which induced their appearance. It is thought that after a second injection of SRBC the IgM-, IgG- and IgA-memory cells can differentiate with the help of the T-memory cells within the bone marrow into IgM-, IgG- and IgA-PFC respectively.The sequence of appearance of the B-memory cells in the bone marrow was shown to be IgM-IgG-IgA.Six months after the intravenous injection of SRBC, the presence of B-memory cells could be demonstrated not only in spleen and bone marrow, but also in peripheral lymph nodes, mesenteric lymph node, Peyer's patches, thymus and blood. The increase in amount of B-memory cells was most prominent in the spleen.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

The origin of antibody-forming cells detected in the bone marrow after thymus-independent antigen-stimulation and abnormality in migration of B cells of X-linked immunodeficient CBA/N mice

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC, response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Antigen-binding cells in central and peripheral lymphoid tissues of the rabbit

The rosette assay was used to study antigen-binding activity by cells in lymphoid tissues of rabbits immunized with sheep red blood cells and in unimmunized controls. Percentages of rosette-forming cells (RFC) observed were compared with those of cells which secreted antibody (plaque-forming cells, PFC) and cells which both bound antigen and secreted antibody. Rosette-forming cells and PFC were shown to be two distinct reactive cell populations. Thus, in the spleen less than 1% of RFC also formed plaques. Immediately following antigen stimulation, the number of RFC in the bone marrow decreased to below detectable limits. After an initial rise, the number of RFC in the appendix declined similarly. In contrast, RFC levels in the spleen rose steadily from the time of immunization. These patterns suggest that bone marrow and appendix may function as a reservoir of antigen-binding cells which are released to other sites following antigenic stimulation. Rosette-forming cells were rarely observed in the thymus. Rosette-inhibition studies using antisera specific for bone marrow-derived cells (anti-B) and thymus-derived cells (anti-T) revealed a markedly greater proportion of T-RFC in the appendix than in the spleen.     

In vitro immune response to the 2,4,6-trinitrophenyl determinant in aged C57BL/6J mice:changes in the humoral immune response to, avidity for the TNP determinant and responsiveness to LPS effect with aging.

An in vitro anti-TNP response of the spleen cells from aged C57BL/6J mice showed approximately 4-fold less PFC than did that from young adult mice. Anti-theta serum-treated young spleen cells gave an anti-TNP response that was definitely greater than the response of the anti-theta serum-treated aged spleen cells in the presence of the exogenous activated thymus cells as helper cells. These results suggest that the deficits in B cells may be partly responsible for the imparied anti-TNP response of the aged spleen cells. To examine further the capacity of stem cells in the bone marrow to generate B cells responsible for anti-TNP response in the spleen, we injected i.v. 1.5 to 2.0 times 10(7) bone marrow cells from young or aged mice into lethally irradiated syngeneic recipients that had previously been thymectomized. Four to 6 weeks later, 10(7) spleen cells from the two groups of these recipient mice were immunized with TNP-SRBC in the presence of the exogenous activated thymus cells and assayed for anti-TNP PFC. The response of the aged marrow-derived B cells was approximately one-half of that of the young marrow-derived B cells.The avidity for TNP determinant of the antibodies produced by the PFC was determined by the plaque-inhibition technique. The avidity of the antibodies produced by the aged mice was approximately 33 times lower than that by the young mice. Anti-TNP response of the young spleen cells were markedly enhanced by the addition of LPS to the cultures, whereas no or little enhancement of the response was induced in the aged spleen cells even in the presence of high concentration of LPS. In contrast, DNA synthesis of both the young and aged spleen cells was comparably stimulated by 1 mug/ml and 10 mug/ml of LPS, however, it was rather less in the aged spleen cells at a concentration of 100 mug/ml. Mechanisms responsible for the changes in avidity and responsiveness to LPS with aging are discussed.     

Changes in the homing properties of labeled lymphoid cells caused by solid tumor growth

The effect of a progressively growing fibrosarcoma upon the distribution of ⁵¹Cr-labeled cells from the lymph nodes, spleen, thymus, bone marrow and Peyer's Patches was measured in tumor-bearing recipient mice. Tumor presence caused a uniform depression of migration of labeled cells to the bone marrow. In most cases increased homing of cells to the spleen was also observed. Labeled cells prepared from lymph nodes and Peyer's Patches were generally unaffected by the presence of a growing tumor. Migration of labeled cells from tumor bearing donors into normal syngeneic recipients suggests depletion or incapacitation of parts of the T-cell population of the spleen. These results emphasize the important relationship between splenic function and tumor progression.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

Studies of the immunological capacity of germfree mouse radiation chimeras. III. In vitro reconstitution of the T-helper cell deficiency

The plaque-forming cell (PFC) response of long-term radiation induced allogeneic bone marrow chimeric (ABMC) mice has been shown to be markedly deficient. The nature of the cellular deficiency of the primary PFC response was investigated using in vitro culture techniques. Adherent spleen cells from ABMC or DBA/2 mice support equally well the development of PFC from nonadherent DBA/2 spleen cells. Nonadherent cells prepared from ABMC mice when cocultivated with DBA/2 adherent cells showed a minimal response. However, the addition of activated DBA/2 T cells to cultures containing adherent cells from DBA/2 mice and nonadherent cells from ABMC mice completely reconstituted the in vitro response to sheep erythrocytes. Therefore a cellular deficiency of the humoral immune system of ABMC mice was shown to be associated with the thymus-derived lymphocyte pool.     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。