A Repopulation Assay For B and T Lymphocyte Stem Cells Employing Radiation Chimaeras

The repopulation of the peripheral lymphoid compartment of lethally-irradiated rats reconstituted with lymphopoietic stem cells was studied. Cell lineages were traced by using genetic markers of cell surface molecules: immunoglobulin allotype for B lymphocytes and peripheral T cell alloantigen for T lymphocytes. Provided the markers had been bred on to a genetic background congenic with the hosts, they conferred neither an advantage nor disadvantage in competition with unmarked cells. the degree of chimaerism measured the lymphopoietic activity of the restorative inoculum. the most potent activity was found in foetal liver and spleen; next was infant spleen and bone marrow; then young adult bone marrow. Peripheral lymphoid tissues showed very little activity and thymus cells were inert. This tissue distribution, the stability of the chimaerism and the substantial expansion of numbers from the injected cells all point to the assay measuring an early stem cell. The overlap of subpopulations of lymphocytes in the rat thoracic duct was studied. A method for the conjugation of fluorescein to antibodies while they are attached to immuno-adsorbent affinity columns is also described.     

Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.     

Partial tolerance and immunity after adoptive abrogation of transplantation tolerance in the rat

Lewis rats were rendered hematopoietic and lymphoid cell chimeras by injection of (LBN)F₁ hybrid cells at birth or following treatment with cyclophosphamide in adult life. The establishment of transplantation tolerance was indicated by acceptance of (LBN)F₁ skin grafts and specific unresponsiveness in graft vs. host reaction (GvHR) and mixed lymphocyte interaction (MLI) in vitro. Tolerance was abolished by adoptively transferred Lewis lymphocytes, and the loss of chimerism and recovery of specific reactivity by blood lymphocytes were monitored independently by mixed lymphocyte cultures. Recovery of competence to initiate GvHR by splenic and lymph node cells was monitored by the local renal graft vs. host technique. Both techniques measure essentially the proliferative response of certain lymphocytes to foreign cellular AgB antigens, and both detected a prolonged, but gradually weakening, state of partial tolerance to the AgB factors to which tolerance had originally been induced. During this phase of partial tolerance the former chimera rejects skin and lymph node cell grafts from (LBN)F₁ donors with alacrity, but in some cases accepts (LBN)F₁ kidney grafts. Cytotoxic antibodies appear in the serum soon after allogeneic chimerism is terminated. These results are interpreted to indicate that a state of partial tolerance exists among the cells which proliferate in response to certain AgB antigens in GvHR and MLI in the formerly tolerant chimera, and that a state of transplantation immunity (possibly to other determinants) coexists with this partial tolerance.     

RNA in the periphery of rapidly proliferating mouse lymphoid cells

RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F₁ or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F₁ mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. ³H-uridine and ¹⁴C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.     

Composition of main haematopoietic compartments in normal and bled channel catfish

Differential cell counts showed that the head and trunk kidney of control and bled channel catfish Ictalurus punctatus had myeloid characteristics. They contained lymphoid and granuloid cells, thrombocytes, erythroid and agranular cells in decreasing order of abundance (%). Among the blast and precursor cells, the most numerous erythroid ones were followed by granuloid, lymphoid and agranular ones. The main changes after blood withdrawal were the decrease of thrombocytes and the increase of precursor cells in both kidney parts. In the group examined 7 days after bleeding the head kidney had a higher percentage of erythroid cells and lymphocyte precursors than the trunk kidney while the latter had more granuloid cells and their precursors. Basophils were present ( c . 1%) in both regions of the kidney of all groups. The spleen was predominantly a lymphatic organ. It contained c . 80% lymphoid cells, a higher incidence of granulated lymphocytes than in kidneys, 15% thrombocytes and 1.4% agranular cells. Blood withdrawal caused an increase of thrombocytes, a decrease of lymphoid cells and an increase of erythroid precursors in the spleen. The last probably stemmed from the circulation. While haematocrit values failed to indicate the anaemic state in the bled groups, the differential red blood cell count showed dramatic differences between the control and bled groups as well as between the two groups in different stages of recuperation from the blood loss.     

Autoimmunity to glial antigens in vitro.

Lymph nodes cells and spleen cells in a 50:50 mixture from Fischer 344 rats were cultured on syngeneic glial cells and fibroblasts. In the glia cultures, but not in the fibroblast cultures, the lymphocytes were stimulated to a vivid blast transformation and mitotic activity with a peak after 7 days, after which they reverted to small- and medium-sized lymphocytes. The stimulated lymphoid cells were not cytotoxic to glial cells when tested in a microcytotoxicity assay. A fraction of the lymphoblasts and their progeny (approximately 4%) took a positive intracytoplasmic stain for γ-globulin immunoglobulin G after direct immunofluorescence. Efforts to demonstrate immunoglobulin produced and released into the culture medium by the stimulated cells were negative. The findings may indicate that among the lymphoid cells responding to the glial antigens under these conditions, there are suppressor cells that abrogate the killer cell effect.     

SPECIFIC positive and negative selection of rat lymphocytes reactive to strong histocompatibility antigens: activation with alloantigens in vitro and in vivo.

This study compares the functional properties of rat thoracic duct lymphocytes (TDL) after stimulation with strong alloantigens of the major histocompatibility complex (MHC) either in vitro in preparative mixed lymphocyte interactions (MLI) or in vivo in systemic graft-vs-host (GVH) reactions. Comparisons were made of PHA responses and reactivity to the specific priming haplotypes or to third party haplotypes in analytical MLI and in GVH reactions either before or after the activated populations were "parked" in syngenetic T cell-deprived (B) rats. These comparisons can be summarized as follows: 1) TDL populations primed in bulk MLI cultures (MLI-TDL) slowed some evidence of specific positive selection when tested immediately; MLI responses to specific alloantigens were both relatively large and accelerated in tempo, whereas responses to third party alloantigens were diminished but also accelerated in tempo. Specific GVH responses were more marked than in third party recipients but they were also decreased relative to normal, and displayed an abberant dose/response slope. MLI-TDL populations tested after they had been stored in syngeneic B rats showed clear evidence of stable-specific positive selection; specific MLI and GVH responses were enriched relative to third party responses and also in comparison to normal, unselected TDL populations. This finding indicates that GVH and MLI reactivity are probably both functional capacities of the same lymphocyte subpopulation since positive selection by one function (MLI) also enriched for a second (GVH). 2) Parental strain TDL activated in vivo in the systemic GVH reaction in irradiated F1 animals and recovered from the thoracic duct 3 to 4 days later (late GVH-TDL) consisted mainly of blast cells, however, in contrast to MLI-TDL these populations showed no evidence of positive selection when tested before or after parking in B rats. MLI responses to specific alloantigens were minimal, and greatly reduced in magnitude compared to normal. GVH responses to specific haplotypes could be detected, but these were not enriched compared to normal, despite the content in the late GVH-TDL populations of a significant proportion of blast cells presumably activated by host alloantigens. 3) Early collections (less than 40 hr) of parental strain GVH-TDL collected from F1 recipients contained no blast cells and showed impressive degrees of negative selection; they were markedly depleted of both GVH and MLI activity to specific alloantigens but displayed normal reactivity to third party alloantigens. Moreover, specific negative selection was persistent in these populations parked for several weeks in B rats, and indication that a specific subpopulation of reactive cells had been physically eliminated. 4) PHA responses of both MLI- and GVH-activated TDL populations tested either before or after parking in B rats were approximately normal on a per T cell basis...     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

Cyclic adenosine 3',5'-monophosphate response to dopamine in mouse lymphoid cells and cell lines

Cyclic adenosine 3',5'-monophosphate responses to dopamine and isoproterenol were studied in mouse and rat spleen, thymus, lymph nodes and Peyer's patches lymphocytes and in 7 mouse cell lines of T- and B-lymphoid derivation. The responses of normal cells to dopamine were moderate, of the same extent, but selective to spleen and thymus in mouse, and to spleen and lymph nodes in rat. The YAC-1 T lymphoma cell line was sensitive to dopamine with a higher magnitude than normal lymphoid cells. Dopamine was less potent than isoproterenol in all cells, and whereas dopamine-sensitive and isoproterenol-sensitive cells, or dopamine-insensitive and isoproterenol-insensitive cells were found, no cell type was dopamine-sensitive and isoproterenol-insensitive. Altogether, these results suggest that only a small subset of lymphocytes is susceptible to the cAMP-elevating action of dopamine.     

Effects of tobacco glycoprotein (TGP) on the immune system. I. TGP is a T-independent B cell mitogen for murine lymphoid cells

It has been shown that tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein antigen purified from cured tobacco leaves, is mitogenic for lymphoid cells in the spleen, peripheral blood, and bone marrow, but not for thymus cells. The proliferative response is not reduced by treatment of spleen cells or peripheral blood lymphocytes with anti-Thy-1.2 and complement, and spleen cells from the congenitally athymic (nu/nu) CD-1 proliferate as vigorously in response to TGP as do spleen cells from their heterozygous nu/+ littermates. In addition, TGP induces differentiation of mouse spleen cells into antibody-secreting cells, the majority of which secrete IgM, and the remainder mainly IgG and a few IgA. The differentiation into antibody-secreting cells induced by TGP occurs with spleen cells from nu/nu mice. It is concluded that TGP is a T-independent B cell mitogen for mouse lymphoid cells. On the basis of the ability of spleen cells from the LPS-nonresponder C3H/HEJ mice to respond to TGP with proliferation and differentiation into antibody-secreting cells, it is concluded that the effects of TGP are distinct from those of LPS and cannot be due to contamination of the TGP preparation with LPS.     

Effects of estrogen on the lymphoid regeneration and immune response in irradiated and marrow-reconstituted mice.

Various doses of estriol (E3) were given to mice intraperitoneally, immediately after lethal irradiation and marrow reconstitution. The assessment of the plaque-forming cell (PFC) response to sheep erythrocytes in the spleen and the histological assessment of lymphoid tissues were carried out 30 days later. The effects appeared to be dose-dependent and resulted in a marked suppression of the PFC response. The depletion of lymphocytes was dramatic and dose-dependent in the thymus, and in the thymus-dependent and in the thymus independent areas of the peripheral lymphoid tissues. These results suggest that E3 acts on the differentiation of stem or precursor cells toweard both the populations of T and B lymphocytes. Although E3, given on day 7 after irradiation and marrow reconstitution, suppressed the lymphoid regeneration and PFC response markedly, E3 given on day 14 had no effect. On day 7 the majority of regenerating lymphoid tissues were large pyroninophilic cells and on day 14, small lymphocytes. These results suggest that the precursor or immature lymphocytes are sensitive to E3, while mature lymphocytes are resistant. Lymphoid regeneration and PFC response were retarded in mice irradiated and reconstituted with bone marrow cells from donors pretreated with E3. These results suggest that E3 acts on the stem or precursor cells capable to differentiate in the direction of lymphoid populations and reduce their number in the bone marrow.     

Characterization of the normal lymphocyte population cytolytic to Burkitt's lymphoma cells of the EB2 cell line.

The normal human lymphocyte population which exhibits "spontaneous" cytolysis of EB2 Burkitt's lymphoma cells has been characterized. The effector cell has EA and EAC' receptors but lacks E receptors and probably surface Ig. "Spontaneous" anti-EB2 cytotoxicity was not reduced by preincubation of the effector cells with plastic or iron carbonyl or by passage through cotton wool or agarose columns but was reduced by passage through nylon wool columns. Thymocytes were not cytotoxic to EB2 cells, and chronic lymphocytic leukaemia cells (of B cell characteristics) had reduced cytotoxicity compared with normal lymphocytes. Cells from various lymphoid organs of rats and guinea-pigs were also cytotoxic to EB2 cells with reactivity in spleen greater than or equal to blood greater than lymph nodes. Spleen cells from neonatally thymectomized rats had increased cytotoxicity compared with normal rat spleen cells, suggesting that T lymphocytes are not essential. The effector cell in rat spleen did not adhere to cotton wool or agarose columns, indicating some resemblance to its counterpart in human peripheral blood.     

3H-uridine incorporation by small lymphocytes of tolerant rats: relationship to T and B lymphocytes.

Tissue tolerance was induced in neonatal rats by the intravenous injection of bone marrow cells from adult allogeneic rat donors. After 6 to 8 weeks, lymphoid cells from rats in which tolerance had been induced were tested for mixed lymphocyte reactivity (MLR), 3H-uridine uptake, and the relationship of uridine incorporation to B and T lymphocytes. Lymph node (LN) and spleen (SPL) cells from the adult inoculated rats showed no reactivity in the MLR or normal lymphocyte transfer reaction (NLTRx), indicating that the animals were tolerant. After in vitro exposure to 3H-uridine, an abundance of small lymphocytes (SL) from these same tolerant rats were heavily labeled, in contrast to nontolerant controls, where relatively few SL were heavily labeled. In order to determine whether the heavily uridine-labeled cells were T cells or B cells, lymphoid cells from the LN and SPL of tolerant animals were exposed to either rabbit anti-AKR brain serum or rabbit anti-rat Ig conjugated with ferritin. The results showed that the heavily uridine-labeled SL of the tolerant rats were mainly Ig-positive cells.     

The Chronic Leukemias: A Review of Disease Manifestations and the Aims of Therapy

Certain aspects of the chronic leukemias that may influence future therapeutic trials are reviewed. In chronic lymphocytic leukemia (CLL), there is minimal mitotic activity in lymphoid tissues; indolent, long-lived lymphocytes, unresponsive to antigenic or phytohemagglutinin (PHA) stimulation accumulate. In many patients, erythroid precursors fail to proliferate despite the stimulus of a severe anemia, but a proliferative response can be initiated by prednisone. We need to know how the normal proliferative responses of these cells are modified, because the correction of these abnormalities would relieve most of the disease manifestations. CLL may not be a neoplastic disorder. In chronic myelogenous leukemia (CML), the leukocyte doubling time shortens as the disease duration lengthens; a significant correlation between this time and survival is demonstrated. Before therapy designed to eliminate the Ph¹-positive (Philadelphia chromosome) stem cell is tried, we need to know whether a normal hematopoietic stem cell exists in Ph¹-positive CML.     

Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

PTA1在大鼠淋巴器官中定位分布的免疫组织化学和原位杂交研究

采用免疫组织化学方法和地高辛－碱酸酶标记原位杂交组织化学方法观察PTA1在大鼠胸腺、脾脏和淋巴结等淋巴器官中的定位分布。首次证实PTA1和PTA1 mRNA散在分布于大鼠脾脏和胸腺中,但在淋巴结中未见分布。本研究结果为全面了解PTA1在体内的分布及功能提供重要的实验依据。     

Retrovirus-mediated modification of male germline stem cells in rats

The ability to isolate, manipulate, and transplant spermatogonial stem cells provides a unique opportunity to modify the germline. We used the rat-to-nude mouse transplantation assay to characterize spermatogonial stem cell activity in rat testes and in culture. Our results indicate that rat spermatogonial stem cells can survive and proliferate in short-term culture, although a net loss of stem cells was observed. Rat spermatogonial stem cells also were susceptible to transduction with a retroviral vector carrying a lacZ reporter transgene. Using a 3-day periodic infection protocol, 0.5% of stem cells originally cultured were transduced and produced transgenic colonies of spermatogenesis in recipient mouse testes. The level of transgenic donor-derived spermatogenesis observed in the rat-to-mouse transplantation was similar to levels that produced transgenic progeny in the mouse-to-mouse transplantation. This work provides a basis for understanding the biology of rat spermatogonial stem cells. Development of an optimal rat recipient testis model and application of these methods for germline modification will enable the production of transgenic rats, potentially valuable tools for evaluating genes and their functions. In addition, these methods may be applicable in other species where existing transgenic methods are inefficient or not available.     

Differences in chromatin thermal lability between thymic and splenic lymphocytes in intact and adrenalectomized rats detected by acridine orange microfluorometry

The sensitivity of chromatin to thermal denaturation was compared between small lymphocytes from the thymus and spleen of intact and adrenalectomized rats. RNA was enzymatically removed from uniformly spread lymphocytes attached to glass slides in low density and chromatin denaturation effected by heating at 90 °C in a solution free of formaldehyde and sodium ions. The preparations were stained with acridine orange following acetylation. Fluorescence emission intensity of the nuclei in individual cells was measured at 530 and 590 nm and the ratio used as a relative index of chromatin denaturation. The data show that the chromatin of small thymus lymphocytes is generally more thermolabile than that of morphologically comparable cells of the spleen in both intact and adrenalectomized animals. This difference between cells of the same morphological type from the two lymphoid organs was not evident from measurements of the amount of dye bound by the cells without denaturation. Removal of the endogenous source of glucocorticoids resulted in an increase in the number of spleen lymphocytes with lower chromatin thermal stability but had no detectable effect on the population of thymus lymphocytes. The results are discussed in terms of the histological organization and immunological role of the thymus and spleen and in relation to the technical aspects of acridine orange microfluorometry as a sensitive cytochemical probe of chromatin structure.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat

Bio-Breeding rat T lymphocytes proliferate poorly in response to alloantigen. Transplantation of Bio-Breeding rats with fetal thymus tissue from diabetes resistant rats leads to an improvement in the T cell proliferative response, but only if the thymus contains bone marrow-derived, radiation-resistant thymic antigen presenting cells of the diabetes-resistant phenotype. The current study provides evidence that thymus transplantation leading to the restoration of Bio-Breeding T cell proliferative function can also significantly reduce the incidence of insulitis and prevent the development of diabetes. It appears that a defect in the bone marrow-derived thymic APC population contributes to an abnormal maturation of Bio-Breeding T lymphocytes which in turn predisposes animals to insulitis and diabetic disease.