Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

The effect of lithium chloride treatment on cell-mediated immunity in mice.

The immunomodulatory effect of lithium chloride (LiCl) administered i.p. to adult mice (150 mg/kg/day) on cellular immunity in vivo was investigated. A short (6-day) treatment with LiCl of either spleen cell donors in semiallogeneic or xenogeneic GVH reaction, or recipients in semiallogeneic HVG reaction, significantly diminished the early sings of local cell-mediated immunity. A short-term LiCl treatment of donor mice also abrogated systemic GVH reaction in newborn F1 recipients, while a treatment prolonged for 20 days did not alter the high GVH response of spleen cells typical for untreated donors. Thus, a striking time-dependent efficacy of LiCl on the reduction of GVH reaction was found.     

Low proliferation capacity of lymphocytes from alloxan-diabetic rats: involvement of high glucose and tyrosine phosphorylation of Shc and IRS-1

The proliferation capacity of lymphocytes obtained from mesenteric lymph nodes of control and alloxan-diabetic (40 mg/kg) rats in response to concanavalin A (ConA) and lipopolysaccharide (LPS) stimuli was examined. Proliferation response of lymphocytes from diabetic rats was significantly reduced under Con A (43%) and LPS (46%) stimulation as compared with the control group. Insulin (166 microM) promoted a marked increase of lymphocyte proliferation (7.5-fold) in the control group and this response was much lower (2.6-fold) in lymphocyte from diabetic rats. Cells were also cultured in medium containing glucose at 5, 10 or 20 mM. High glucose concentration (20 mM) caused a marked inhibition of lymphocyte proliferation reaching the values of the diabetic group. In lymphocytes from control rats, the degree of Shc tyrosine phosphorylation was gradually increased, whereas that of cells from diabetic rats was much lower in response to insulin. In lymphocytes obtained from control rats, the tyrosine phosphorylation of IRS-1 was time-dependent on insulin. In cells from diabetic rats, the basal tyrosine phosphorylation of IRS-1 was higher than that of control rats, however, there was no further phosphorylation after insulin addition. We conclude that the response of lymphocyte proliferation from diabetic rats to Con A and LPS stimuli is decreased but insulin was able to promote a significant proliferative effect on these cells. Also, high glycemia in addition to the lack of insulin participates in the reduced proliferation capacity of lymphocytes from diabetic rats.     

Hematopoietic factors in graft-versus-host reaction

Graft-versus-host (GVH) reaction has a curious unsolved area in the immunopathogenesis and pathophysiology of the immunohematopoietic system, and GVH disease remains one of the major obstacles in clinical allogeneic bone marrow transplantation. T lymphocytes and T lymphocyte subpopulations are now recognized to be initiators of this GVH reaction and disease. Also, T lymphocytes are known to be accessory cells in the regulation of hematopoiesis, and produce a variety of lymphokines relevant to hematopoiesis. Admittedly, remarkable hematopoietic changes can be found in GVH reaction, but the cellular mechanisms underlying these changes are so complex they have yet to be fully elucidated. In fact, elevated serum levels of myeloid and erythroid colony-stimulating activities were found in mice suffering from GVH disease in which marked granulopoiesis and suppression of erythropoietic differentiation were seen. In addition, each granulocyte/macrophage colony-stimulating factor (GM-CSF) or burst-promoting activity (BPA) could be detected in sera from patients with GVH disease following allogeneic bone marrow transplantation. There seems to be at least two mechanisms involved in the control of hematopoiesis with either humoral or local environmental factor, probably via the T lymphocytes or T lymphocyte subpopulations activated by alloantigens or autologous non-T cells.     

Disturbance of lymphocyte circulation byPseudomonas aeruginosa infection in oxazolone-sensitized mice

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone in mice. To test whether an altered lymphocyte circulation plays a role in this depression⁵¹Cr-labeled lymphocytes fromP. aeruginosa-infected and oxazolone-sensitized donors were injected intravenously into infected and sensitized recipients, and the radioactivity uptake of several organs was determine. The controls consisted of normal mice receiving labeled lymphocytes from normal donors. While the radioactivity recovered from the liver, spleen, and mesenteric lymph nodes was similar in the test and the control group, significantly more radioactivity was recovered from the draining lymph nodes of infected and sensitized recipients. The concentration of labeled lymphocytes from sensitized donors in the draining lymph nodes of sensitized recipients was 18% greater than that of the controls but 31% lower than that of infected and sensitized animals receiving cells from infected and sensitized donors.P. aeruginosa infection enhances lymphocyte entrapment within the draining lymph nodes of oxazolone-sensitized mice.     

Effects of lymphocytes from the thymus and lymph nodes on differentiation of hemopoietic spleen colonies in irradiated mice

Hemopoietic colonies were counted macroscopically and microscopically in spleens of hybrid mice seven or eight days after they had been irradiated and given parental bone marrow in donor-host combinations exhibiting poor growth. Colonies counted microscopically were classified as to differentiation pathway. Lymphocytes from the thymus or lymph nodes were injected into some recipients at several different dosages and lymphocyte: bone marrow (L:B) ratios. In confirmation of earlier work it was found that thymocytes increased the number and size of colonies in recipients of marrow. A shift of differentiation toward granulopoiesis was also seen when thymocytes were given, although erythropoietic colonies were still the most frequently seen type except at very high L:B ratios. Lymph node lymphocytes shifted the pattern more markedly toward granulopoiesis, even at low L:B ratios. When lymphocytes from either source were given without marrow, only a few colonies could be found in recipients, and if differentiated they were almost exclusively granulopoietic. Irradiation (900 R) of lymphocyte donors reversed the shift so that a normal pattern of differentiation, like that resulting from marrow alone, was seen; irradiated lymphocytes were nonetheless capable of augmenting the size and total number of hemopoietic colonies.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.     

Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

Both the Lyt-2+ and L3T4+ T cell subsets are required for the transfer of diabetes in nonobese diabetic mice

The nonobese diabetic mouse is a model of spontaneous type I diabetes mellitus. It is possible to induce diabetes in young, irradiated nonobese diabetic mice by using adoptive transfer of splenocytes or splenic T cells obtained from diabetic donors. This study demonstrates that the induction of diabetes in the adoptive transfer system is dependent on both the L3T4+ and Lyt-2+ subsets of T cells. Neither of these T cell subsets alone mediates the development of severe insulitis or diabetes when adoptively transferred to young, irradiated recipients. In addition, we show that both the L3T4+ and Lyt-2+ subsets must be obtained from diabetic donors in order to transfer diabetes; neither subset can be replaced with cells obtained from young, nondiabetic donors.     

Adult thymectomy promotes the manifestation of autoreactive lymphocytes.

Spleen cells from adult thymectomized mice (ATX) were assayed in a syngeneic graft vs host (GVH) model based upon enlargement of the draining popliteal lymph node following syngeneic cell inoculation into the hind footpad. Spleen cells from ATX mice have been found to induce a significantly higher increase in the weight of the regional lymph node than that induced by the injection of normal spleen cells. Irradiated spleen cells from ATX donors did not cause a similar increase, suggesting either that proliferation of the transferred cells was required at some stage of the reaction or that autoreactive cells are radiosensitive. Autoreactive cells were found in the spleen of mice 2 to 3 months after the thymectomy but were never found in the lymph nodes of such animals or in the thymus of intact mice. They are not phagocytic adherent cells and are not retained on nylon wool columns, which suggests that they belong to the T-cell lineage. Autoreactivity is lost when spleen cells from ATX donors are depleted of autologous rosette-forming cells (A-RFC) by centrifugation on a Ficoll-Hypaque gradient after rosette formation. Autoreactive spleen lymphocytes might belong to the population of A-RFC previously characterized as a population of immature T cells.     

Effect of the transplantation of heterochronic thymus on the development of immunological reactivity in CBA mice. 2. Transplantation to intact animals

The effect of transplantation of thymus from donors of different age on the immune system development was studied in the newborn CBA mice. The thymuses of 22 month and 5 day old donors were shown to exert different effects: accelerated (early attainment of the plateau) and decelerated (late attainment of the peak) maturation of the immune system in the recipients. This fact is discussed with respect to interaction of transplanted and host thymuses.     

Alterations in the responsiveness of diabetic fibroblasts to insulin

Fibroblastic cultures from the skin of nondiabetic and diabetic (db/db) mice have been used to investigate alterations in the biological responses of diabetic cells to insulin. Confluent cultures from the skin of both nondiabetic and diabetic animals possess specific receptors for insulin. Diabetic fibroblasts exhibit only 36% as much specific binding of insulin as nondiabetic fibroblasts, because of a decrease in the total number of binding sites, without a change in binding affinity. Insulin caused a time- and dose-dependent increase in the rate of 2-deoxy D-glucose (dGlc) uptake and in ornithine decarboxylase (ODC) activity of both nondiabetic and diabetic fibroblasts. In nondiabetic cells, half-maximal increase in dGlc uptake was obtained with 0.3 nM insulin, and a maximum increase of 120% was obtained with 4.1 nM insulin. In contrast, diabetic cultures required 0.8 nM insulin for a half-maximal increase in dGlc uptake, and maximum stimulation with 4.1 nM insulin was only 50% above control levels. With 4-fold higher insulin concentrations, ODC activity of diabetic cells was only 40% that of nondiabetic cells. In nondiabetic cells, down regulation of insulin receptors by insulin abolished the ability of insulin to stimulate dGlc uptake. These results demonstrate that cells cultured from diabetic animals, which possess a decreased number of insulin receptors, also exhibit decreased stimulation of deoxy D-glucose uptake and ornithin decarboxylase activity by insulin.     

Lymphocyte accumulation in the spleen of retinoic acid receptor-related orphan receptor gamma-deficient mice

The hormone nuclear receptor retinoic acid receptor-related orphan receptor gamma (RORgamma) plays important roles in thymocyte development and lymphoid organogenesis. RORgamma and its thymus-specific isoform RORgammat are expressed in the thymus, but not in the spleen and bone marrow (BM). However, RORgamma(-/-) mice have 2- to 3-fold more splenocytes than wild-type controls due to an accumulation of conventional resting B lymphocytes. The increase in B lymphocytes in RORgamma(-/-) mice is caused neither by abnormal B cell development in the BM nor by an obvious defect in the peripheral T cell compartment. Furthermore, analyses of BM chimeras using either RORgamma(-/-) or recombinase-activating gene-2(-/-) mice as recipients and wild-type or RORgamma(-/-) mice as donors, respectively, demonstrate that the splenic microenvironment of RORgamma(-/-) mice is defective, since wild-type T and B lymphocytes accumulated in these chimeric mice. In addition, T lymphocyte homeostasis was altered due to a lowered thymic output in RORgamma(-/-) mice. Collectively, these results suggest that RORgamma regulates lymphocyte homeostasis at multiple levels.     

Role of cytotoxic T lymphocytes in the prevention of lupus-like disease occurring in a murine model of graft-vs-host disease

The inoculation of B6D2F1 mice with T lymphocytes from the C57BL/6 parental strain induces an "immunosuppressive" graft-vs-host reaction (B6 GVH), whereas inoculation of T cells from the other, DBA/2 parental strain induces an "immunostimulatory" GVH reaction and a lupus-like disease (DBA GVH). The present study compares cytotoxic T lymphocyte (CTL) function in the spleens of these GVH mice as well as differences in the donor inoculum that could account for these different types of GVH. We observed that the B6 GVH induces an immunodeficiency that encompasses CTL precursors (and possibly T helper cells) and results in suppressor cells that abrogate responses to both trinitrophenyl (TNP)-modified self and third party alloantigens. In contrast, the DBA GVH induces only a T helper cell immunodeficiency and results in suppressor cells selective for class II restricted L3T4+ T helper cells. Chimeric T cells were detected in both types of GVH. In the B6 GVH both L3T4+ and Lyt-2+ donor cells were observed, although Lyt-2+ cells predominated. In the DBA GVH, donor T cells were almost exclusively of the L3T4+ phenotype. The lack of appreciable donor Lyt-2+ cells in the DBA GVH can be explained by a defect in the DBA donor inoculum manifested by a naturally occurring two-fold reduction in Lyt-2+ cell numbers as well as a nine-fold reduction in CTL precursors with anti-F1 specificity. T cells in the DBA inoculum, therefore, are predominantly L3T4+. A similar defect induced in B6 donor cells by anti-Lyt2 antibody and complement not only converted the suppressive GVH to a stimulatory GVH, as measured by anti-DNA antibodies, but also resulted in a T cell immune deficiency characteristic of the DBA GVH, i.e., a selective loss of the TNP-self CTL response. Thus the presence or absence of adequate numbers of functioning Lyt-2+ cells in the donor inoculum is correlated with the development of either a suppressive or stimulatory GVH, respectively. That donor Lyt-2+ cells mediate a suppressive GVH through cytolytic mechanisms is evidenced by greater than 70% reduction in B6 GVH spleen cell numbers and readily demonstrable anti-F1 CTL activity by these spleen cells despite an inability to generate anti-allogeneic or anti-TNP self CTL activity even in the presence of added T helper factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental allergic encephalomyelitis: activation of suppressor T lymphocytes by a modified sequence of the T effector determinant

Deletion of certain amino acid residues from the amino acid sequence of the encephalitogenic determinant for guinea pigs, H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, destroyed its ability to induce experimental allergic encephalomyelitis (EAE), a cell-mediated autoimmune disease of myelin. The administration of the modified determinant in the form of 4 repeating pentameric sequences, H-(Phe-Ser-Trp-Gln-Lys)4-Gly-OH, activated an antigen-specific T suppressor lymphocyte subset that rendered both presensitized donors and recipients of donor T lymphocytes unresponsive to an encephalitogenic challenge. Treatment of donors or recipients with cyclophosphamide before or after lymphocyte transfer, respectively, obliterated the ability of peptide S42-sensitized T lymphocytes to induce a state of unresponsiveness to an EAE-challenge. The results establish the existence of antigenic determinants for both immunoinduction and immunoregulation of EAE. The immunoregulatory determinant that activates antigen-specific and cyclophosphamide-sensitive suppressor T lymphocyte subset is sequestered within the disease-inducing or T effector determinant.     

Antigenic markers on mouse lymphoid cells: origin of cells mediating immunologic memory

Specific antisera were used for the purification of thymus dependent and thymus independent or bursa equivalent lymphoid cells in the mouse. Spleen cells from mice immune to sheep erythrocytes, a thymus dependent antigen, or to E. coli 055:B5 lipopolysaccharide, a thymus independent antigen, were treated with anti-θ (C3H) serum or anti-MBLA serum and complement prior to their adoptive transfer into lethally irradiated syngeneic recipients. Syngeneic thymocytes, bone marrow cells, or spleen cells from nonimmune donors were appropriately added to antiserum treated cells prior to transfer. The secondary response to these antigens was assayed in recipient spleens six days after cell transfer. The kinetics of the primary response to SRBC was investigated as to its effect on origin of specific hyper-reactive T or B lymphoid cells.The adoptive response to CPS originated in the B lymphoid cell population. Immunologic memory to CPS was demonstrated in recipients of immune cells, compared to recipients of normal cells, by a five fold increase in antibody forming cells.The IgM and IgG adoptive immune response to high doses of SRBC depended upon an increased number of specifically hyper-reactive T-lymphoid cells to facilitate cooperation between T and B lymphocytes. High doses of SRBC initially stimulated T cell memory but at 42 days after priming an increased number of specifically hyper-reactive B lymphoid cells were present.     

杨树桑黄饮片水提物对免疫低下小鼠的免疫功能的影响

探究桑黄饮片水提物对环磷酰胺所致免疫低下小鼠的免疫功能的影响。本研究以桑黄饮片为原料,采用超声和沸水提取方法得到桑黄饮片水提物,注射环磷酰胺造成小鼠免疫功能低下,通过测定脾脏和胸腺指数、血清免疫球蛋白和细胞因子、脾淋巴细胞增殖、碳粒廓清等实验,对免疫低下小鼠的免疫功能进行研究评价。结果表明,灌胃桑黄饮片水提物的小鼠与模型组相比,免疫器官指数、血清中免疫球蛋白含量、细胞因子水平、脾淋巴细胞增殖程度、巨噬细胞的吞噬速率和吞噬指数、小鼠耳肿胀度均有提高,脾脏苏木精-伊红(HE)染色也表明桑黄饮片水提物可以改善环磷酰胺导致的脾脏组织病理状态。本研究表明桑黄饮片水提物对环磷酰胺所致的免疫抑制小鼠具有良好的免疫保护作用,为桑黄饮片扩大临床应用提供实验依据。     

Regulation of bone marrow lymphocyte production: IV. Cells mediating the stimulation of marrow lymphocyte production by sheep red blood cells: Studies in anti-IgM-suppressed mice,athymic mice,and silica-treated mice

Some cellular requirements have been examined for the stimulation of lymphocyte production in mouse bone marrow by injected sheep red blood cells (SRBC). The increased genesis of marrow lymphocytes after a single dose of SRBC assayed radioautographically after [³H]thymidine labeling was unimpaired in the marrow of mice treated with anti-IgM antibodies from birth to eliminate B lymphocytes, and in congenitally athymic mice lacking T lymphocytes. However, pretreatment of mice with silica to depress macrophage function completely abolished the SRBC effect both on the total lymphocyte production and on the number of B and null small lymphocytes in the marrow. Comparative studies were performed on the thymus and spleen. The results demonstrate that the stimulation of marrow lymphocyte production by SRBC is mediated by a silica-sensitive mechanism, does not require B or T lymphocytes, and is independent of the humoral immune response. Thus, extrinsic agents may amplify the production of primary B cells and other lymphocytes in the bone marrow by an antigen-nonspecific mechanism, putatively mediated by macrophages.     

Some endocrine aspects of the thymus gland.

In studies of the mouse thymus, lymphocyte mitoses are seen to be most frequent in the thymus cortex. There is evidence from thymic grafts that a hypothetical factor, thymopoietin, may stimulate mitosis of thymic lymphocytes. It is a factor which is postulated to act in conjunction with the PAS-positive mesenchymal reticular cells and epithelial reticular cells of the cortex. The thymus medulla is necessary for the integrity of thymic grafts, and may also elaborate a secretion for maintaining the cellular functions of the gland. Thymectomy has been used as a gauge for judging normal thymic function and results, in the mouse, in lymphopenia, degeneration of spleen and lymph nodes, delayed rejection of skin allografts, reduced ability of spleen cells to mount the graft versus host reaction, and reduced primary immune response to certain antigens. Correction of these deficiencies offers a means of evaluating various thymic extracts and grafts. Lymphocytosis-stimulating hormone (LSH) is known to maintain the peripheral lymphoid organs and cause lymphocytosis in the thymectomized animal. Diffusion chamber studies of thymic grafts also show restored lymphoid tissue by a cell-free factor (CIF). These two factors may be the same and probably represent the basis of the highly purified lymphocyte-stimulating proteins, LSHr and LSHh, which restore the L/P ratio in thymectomized animals and may stimulate lymphopoiesis in spleen and lymph nodes. LSHr, unlike LSHh, increases the total lymphocyte count. LSHr has been found to increase the humoral antibody response in neonatal mice both by the PFC technique and by direct hemolysis of sheep erythrocytes. Homeostatic thymic hormone (HTH) is a thymic extract of small molecular weight and contains nucleic acid. In the thymectomized guinea pig it has been found to maintain normal levels of lymphocytes in the blood, spleen and lymph nodes, to restore antibody titers to typhoid H antigen and to restore the toxic allergic reaction. Thymic humoral factor (THF) is of smaller molecular weight (less than 1,000) and probably is not a protein. It also enhances lymphoid proliferation in neonatally thymectomized mice. There is evidence that THF participates in humoral antibody formation because it stimulates PFC formation from neonatally thymectomized mice after inoculation with sheep erythrocytes. Its effects on cell-mediated immunity are seen from findings that injection of THF restores the ability of thymectomized mice to reject skin allografts. THF enables spleen cells from thymectomized or neonatal animals to mount the graft versus host reaction, and causes maturation of bone marrow cells and spleen or lymph node cells so that they can participate in the graft versus host reaction. It has been reported to stimulate lymphocytes to kill isogeneic tumor cells in vitro. Thymosin is protein extracted from the thymus. It has been found to alleviate leukopenia slightly and provide some improvement in lymphoid histology in thymectomized mice...