Influence of thymectomy on the lymphoid regeneration of hematopoietic bone marrow after sublethal irradiation]

In C57BL mice, bone marrow lymphoid regeneration after a sublethal irradiation is modified by a graft of normal marrow cells. This effect is suppressed in thymectomized mice since a lymphoid peak is observed after a 350 R irradiation; its composition is heterogeneous: small lymphocytes, lymphoblasts and peculier cells named "X cells". The same phenomenon is observed in mice where all the thymocytes and thymus derived and peripheral lymphocytes are destroyed. These results exclude that bone marrow lymphoid regeneration after irradiation is due to a migration of lymphoid cells of thymic origin to the marrow. They could be explained by the effect of a humoral thymic factor on marrow lymphopoiesis.     

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.     

Quantitative evaluation of the total number and distribution of lymphocytes in young pigs.

In young pigs, the spleen, thymus and all lymph nodes were dissected out and weighed. The relative content of lymphoid cells was determined from histological sections. The number of nucleated cells was evaluated by two different methods: firstly, by measuring the DNA content of samples of lymphoid tissue and dividing by the DNA content of a single nucleus; and, secondly, by counting all lymphoid cells in histological sections of defined volumes of these organs. The number of lymphoid cells in tonsils, gut, bone marrow and lung were determined using histological evaluations and the volumes or weights of these organs. The resulting average number of lymphocytes was 321 times 10 (9) for a pig of 26 kg body weight. The lymphocytes showed the following distribution in lymphoid and non-lymphoid organs: thymus 44%, spleen 9%, mesenteric lymph nodes 17%, cervical lymph nodes 9%, other peripheral lymph nodes 3%, gut-associated lymphocytes 5%, tonsils 2%, bone marrow 5%, blood 3%, lung 0.2% and an estimated figure of 3% for all other tissues.     

The role of the thymus in the ontogeny of the immune system

The proper development of the organs of the immune system is dependent on at least three factors: (1) the development of anlagen with the capacity to trap antigens and support the proliferation of lymphoid and plasma cell precursors; (2) the production by the bone marrow of lymphoid and plasma cell precursors which seed in the lymphoid organs; and (3) the thymus, which seeds reactive cells to the lymphoid organs and produces a humoral factor stimulating antigen-triggered proliferation of primitive lymphoid and plasma cells. Studies on cell population changes in the lymph nodes following thymectomy in mice confirm earlier evidence that most cells produced in the thymus do not seed to the lymphoid organs, but die locally in the thymus.     

Ultradian Rhythms of Tumor Resistance System Activity

Ultradian time-dependency of the carcinogenic effect was studied in mice; both the model of transplanted (leukemia EL-4) and that of chemically-induced (urethane-induced lung adenomas) tumor were used. As the possible mechanisms which are attributable to this phenomenon, ultradian variations in cellularity of the lymphoid organs (thymus, lymph nodes, spleen, bone marrow), in natural killer-cell (NK) activity, level of the serum factors modifying lymphocyte migration, and in activity of the liver carcinogen-metabolizing enzyme system were investigated. All these characteristics were determined hourly in intact mice. Both the life span of mice inoculated with tumor cells, and the yield of carcinogen-induced tumors were found to be ultradian dependent (periods 3-8 hr). Ultradian dependency was found also for the number of lymphocytes in the lymphoid organs, the number of karyocytes in bone marrow, the NK activity against tumor cells, the migration index of normal spleen cells in the presence of tumor cells, as well as for the activity of the carcinogen-metabolizing enzyme system. It is suggested that variations in the natural resistance against transplanted or chemically-induced tumors may be due to the variations in these parameters.     

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.     

The effect of haemorrhage on the cell populations of the thymus and bone marrow in wild starlings (Sturnus vulgaris)

Summary Following the withdrawal of blood from the brachial vein of adult wild starlings (Sturnus vulgaris) changes in the cell populations within the bone marrow and thymus were observed over an eight day period. The packed cell volume, haemoglobin content and reticulocyte count of the peripheral blood was determined before and after haemorrhage.The maximum effect of the haemorrhage was observed in the bone marrow after four days when the population of small lymphocytes, and basophilic erythroid precursors were reduced to less than 1%. At the same time the percentage of another line of erythroid cells increased to 68%. This second erythroid lineage was the major erythroid line in the thymus, and again maximum representation occurred at 4 days post haemorrhage. After this the thymus became predominantly lymphoid and started to increase in size.The two erythroid lines are described and their status with regard to avian thrombocytes is also discussed.The peripheral blood had not attained the pre-haemorrhagic values for reticulocyte counts by eight days although the packed cell volumes and haemoglobin contents were similar.I would like to thank Dr. Peter Ward of the Institute of Terrestrial Ecology for help in obtaining the starlings. Thanks are also due to the staff of the Anatomy Department of St. Thomas's Hospital Medical School, and in particular Mr. Watson. This and other work on the thymus is possible due to the support of the Research (Endowments) Committee of St. Thomas's Hospital     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Heterogeneity of bone marrow lymphocytes: radioautographic detection of pre-B cells bearing cytoplasmic mu chains, and of B and T lymphocytes, and characterization of null lymphoid cells

A radioautographic immunolabeling technique has been developed to detect pre-B cells bearing cytoplasmic mu chains among populations of bone marrow lymphoid cells identified by conventional hematologic stains. 125I-Anti-mu antibody was applied either to fixed marrow smears, labeling total mu chains both in the cytoplasm (c mu) and at the cell surface (s mu), or to cell suspensions, labeling s mu alone. In stained radioautographs the incidence of c mu+ s mu- pre-B cells was derived both indirectly by subtracting values for s mu+ cells from those for total mu+ cells of various sizes in normal mice and directly by the total mu chain labeling in mice depleted of s mu+ cells by anti-IgM treatment in vivo. Binding specificity was demonstrated by the displacement of labeling by nonradioactive anti-mu antibody. The c mu+ s mu- cells showed a bimodal size distribution. They accounted for 40% of the large lymphoid cells and 30% of the small lymphocytes in the marrow. A further 50% of the small lymphocytes were B lymphocytes (s mu+) and 8% were T lymphocytes (Thy 1.2+). Thus, the technique demonstrates the presence of c mu+ s mu- pre-B cells among both proliferating large lymphoid cells and nondividing small lymphocytes, as classically defined in marrow smears. In addition, the results reveal a broad size distribution of mu- lymphoid cells, including a subset of small lymphocytes which lack c mu, s mu, and Thy 1.2 and thus cannot be assigned to either B or T lineage by these criteria. The findings suggest that in addition to B cells the marrow may produce other types of lymphoid cells, yet to be defined.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

Immunomodulating action of myelopeptides in severe closed experimental craniocerebral injury in rats

An immunocorrective effect of myelopeptides (MP) isolated from pig bone marrow cell culture supernatant in the early posttraumatic period in rats with severe cranial injury has been assessed. MP administration prevented cellular devastation of thymus and bone marrow, as well as spleen hyperplasia. The most marked MP effect was observed within the first 24 h after its administration. MP affected the functional and migration properties of both the entire population of lymphoid cells and individual subpopulations. MP had a pronounced protective effect against Staph. aureus infection during cranial trauma. MP completely prevented the death of animals and reduced more than 3-fold Staph. aureus persistence in the organism of animals. Anti-stress and protective effects of MP open vast prospects for their therapeutic and preventive application in the clinical practice.     

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.     

Bone marrow-derived hemopoietic precursors commit to the T cell lineage only after arrival in the thymic microenvironment

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.     

Post-irradiation thymocyte regeneration after bone marrow transplantation. II. Physical characteristics of thymocyte progenitor cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H x AKR)F1 bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, the thymocyte progenitor cells show the same distribution as CFUs. The relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Studies of the cellular cure for osteopetrosis by transplanted cells: specificity of the cell type in ia rats.

Spleen cells from normal rats are known to cure osteopetrosis in ia littermates within 3 weeks. In this study cell suspensions from liver, thymus, bone marrow, salivary gland, skeletal muscle and brain from normal rats were tested for their ability to cure osteopetrosis in ia littermates whose ability to reject these cells had been suppressed by whole-body irradiation. Cells from liver, thymus and bone marrow cured the disease as effectively as spleen cells from normal littermates. Mutants that received cells from salivary gland, muscle and brain remained osteopetrotic. These data suggest that some cell found in spleen, liver, thymus and bone marrow of 10-day-old normal rats, such as a lymphoid cell or stem cell, can restore hemopoiesis and bone resorption in osteopetrotic (ia) rats.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

MIGRATION OF SMALL LYMPHOCYTES IN ADULT MICE DEMONSTRATED BY PARABIOSIS

Parabiotic BALB/C mice were used to study the traffic of small lymphocytes in immunological mature but unchallenged mice. By giving ³H-thymidine (³H-TdR) injections to only one member (A) of a pair by preventing the escape of the radioactive isotope to the other member (B), the kinetics of newly-formed cells was followed. Less than 10% labelled small lymphocytes were found in the peripheral lymphoid tissues of both A and B members, while the thymusses and bone marrows of A members showed labelling percentages up to 70% in this period. Hardly any labelled cells gained entrance into the thymus while a detectable number was found in the bone marrows of B members. Results from pairs set up to follow migration of long-lived lymphocytes revealed that labelled cells detected 4–5 weeks after injections were equilibrated between the peripheral tissues and the bone marrows of the partners. Very few labelled cells were seen in the thymic medulla and none were observed in the thymic cortex, germinal centres or medullary cords of lymph nodes from any B member. It was concluded that short-lived small lymphocytes are formed primarily in the thymus and bone marrow and the migration of these cells is limited in adult animals. Furthermore, the vast majority of long-lived small lymphocytes are freely recirculating, and these cells gain entrance to and are normal residents in the bone marrow.     

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.