Antigenic relationship between bone marrow lymphocytes cortical thymocytes and a subpopulation of peripheral T cells in the rat: description of a bone marrow lymphocyte antigen.

Populations of rat bone marrow lymphocytes (BML) consisting of approximately 90 percent, “tnull” cells were prepared by density gradient centrifugation, passage through a column of fine glass beads, and treatment with anti-T cell and anti-B cell serum plus complement. Antisera to these bone marrow lymphocytes were raised in rabbits. After absorption with RBC and peritoneal exudate cells, the anti-BML sera were found by immunofluorescence to react selectively with “null” cells in bone marrow, with cortical thymocytes, and with a cortisone-sensitive subset of T cells in blood and in spleen, possibly in red pulp. The antigen that is common to these cell types is designated the rat bone marrow lymphocyte antigen (RBMLA). Lymphocytes that are positive fur KBMLA are negative for another lymphocyte-specific heteroantigen, rat musked thymocyte antigen (RMTA). As shown previously, RMTA is present on medullary thymocytes and ou cortisone-resistant T cells in white pulp of spleen, paracortex of lymph node and thoracic duct lymph. It is postulated that two developmentally and functionally distinct lines of T cells exist in peripheral lymphoid tissues of the rat, one derived from cortical thymocytes and one derived from medullary thymocytes. It is further postulated that the “null” population of bone marrow lymphocytes contains the lymphopoietic stem cells from which these two lines of T cells originate.     

Evidence for the existence of multipotential lympho-hematopoietic stem cells in adult rat

Rats were given near-lethal doses of x-ray to produce clones of hemic cells marked by radiation-induced chromosome abnormalities. Subsequently, bone marrow from these rats was injected into lethally irradiated mice to form erythropoietic spleen colonies; and peripheral blood lymphocytes from the same rats were stimulated to proliferate in a mixed lymphocyte interaction (MLI), an immunological response to histocompatibility isoantigens. Chromosome markers indicated that in several instances the cells of an erythroid spleen colony and a proportion of the lymphocytes reacting in the MLI were progeny of the same stem cell in the donor rat. In addition, lymphocytes of the same radiation-marked clone were shown to proliferate in response to several different histocompatibility isoantigens. The data indicate the presence in the adult rat of a primitive lymphohematopoietic stem cell capable of yielding both erythroid and lymphoid progeny. The findings also suggest that immunological specificity is determined during lymphoid differentiation, subsequent to the stem cell stage.     

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.     

The effect of an antigenic stimulation on proliferative response in athymic mice

The proliferation activity of the main cellular categories of bone marrow after infusion of³H-thymidine was studied in nu/nu and +/+ 1-month- and 3-month-old BALB/c mice in comparison with lymphoid cells in the spleen and mesenteric lymph nodes. The stem cell defect in nu/nu mouse bone marrow is compensated by an, increased proliferation in myeloid series and in agranulocytes. The increase of proliferation activity among lymphoid cells in peripheral lymphoid organs was observed only in the 3-month-old mice with a delay in the nudes.     

Self-maintenance of migrating hematopoietic stem cells]

Decreased self-maintenance ability of the migrating stem cells (CFU) from the peripheral blood or ectopic focus of hemopoiesis in comparison to the settled bone marrow CFU, as measured by the spleen colony method or by means of chromosomal markers, has been studied. The competence for myeloid and lymphoid differentiation was essentially the same for migrating and settled stem cells.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Effect of selective T cell depletion of host and/or donor bone marrow on lymphopoietic repopulation, tolerance, and graft-vs-host disease in mixed allogeneic chimeras (B10 + B10.D2----B10)

Reconstitution of lethally irradiated mice with a mixture of T cell-depleted syngeneic plus T cell-depleted allogeneic bone marrow (B10 + B10.D2----B10) leads to the induction of mixed lymphopoietic chimerism, excellent survivals, specific in vivo transplantation tolerance to subsequent donor strain skin grafts, and specific in vitro unresponsiveness to allogeneic donor lymphoid elements as assessed by mixed lymphocyte reaction (MLR) proliferative and cell-mediated lympholysis (CML) cytotoxicity assays. When B10 recipient mice received mixed marrow inocula in which the syngeneic component had not been T cell depleted, whether or not the allogeneic donor marrow was treated, they repopulated exclusively with host-type cells, promptly rejected donor-type skin allografts, and were reactive in vitro to the allogeneic donor by CML and MLR assays. In contrast, T cell depletion of the syngeneic component of the mixed marrow inocula resulted in specific acceptance of allogeneic donor strain skin grafts, whether or not the allogeneic bone marrow was T cell depleted. Such animals were specifically unreactive to allogeneic donor lymphoid elements in vitro by CML and MLR, but were reactive to third party. When both the syngeneic and allogeneic marrow were T cell depleted, variable percentages of host- and donor-type lymphoid elements were detected in the mixed reconstituted host. When only the syngeneic bone marrow was T cell depleted, animals repopulated exclusively with donor-type cells. Although these animals had detectable in vitro anti-host (B10) reactivity by CML and MLR and reconstituted as fully allogeneic chimeras, they exhibited excellent survival and had no in vivo evidence for graft-vs-host disease. In addition, experiments in which untreated donor spleen cells were added to the inocula in this last group suggest that the presence of T cell-depleted syngeneic bone marrow cells diminishes graft-vs-host disease and the mortality from it. This system may be helpful as a model for the study of alloresistance and for the identification of syngeneic cell phenotypes, which when present prevent engraftment of allogeneic marrow.     

The T-cell receptor: just another hypothesis.

Functional activities of T and B lymphocytes and the kinetics of hematopoietic stem cells were studied in mice with inoculated or spontaneous tumors. The development and growth of the tumor inhibited B cells and helper T cells, while the activity of killer T cells and spleen suppressor cells was markedly enhanced. The processes of stem cell migration from the bone marrow were considerably intensified and altered in tumor-bearing mice. Data were obtained suggesting that helper T cells and killer T cells represent nonidentical compartments within the population of thymus-dependent lymphocytes. Immunosuppression during tumor bearing is probably due to an impairment of T lymphocytes cooperating in immune responses, B-lymphocytes and their precursors.     

Impaired development of T lymphoid precursors from pluripotent hematopoietic stem cells in New Zealand Black mice.

Bone marrow cells from autoimmune-prone New Zealand Black (NZB) mice are less efficient at colonizing fetal thymic lobes than cells from normal strains. This study demonstrates that the reduced capacity of NZB bone marrow cells to repopulate the thymus does not result from their inability to migrate to or enter the thymus. Rather, the T lymphopoietic defect of NZB mice is due to an impaired ability of pluripotent hematopoietic stem cells (PHSCs) to generate more committed lymphoid progeny, which could include common lymphoid precursors and/or other T cell-committed progenitors. Although PHSCs from NZB mice were not as efficient at thymic repopulation as comparable numbers of PHSCs from control strains, the ability of common lymphoid precursors from NZB mice to repopulate the thymus was not defective. Similarly, more differentiated NZB T cell precursors included in the intrathymic pool of CD4(-)CD8(-) cells also exhibited normal T lymphopoietic potential. Taken together, the results identify an unappreciated defect in NZB mice and provide further evidence that generation of lymphoid progeny from the PHSCs is a regulated event.     

Effect of exposure to stress on the role of T- and B-lymphocytes in the response of the hematopoietic system

Cellular composition of the bone marrow, spleen and peripheral blood was studied after 6-hour immobilization on the back in experiments on 4 groups of (CBAxC57BL)F1 mice with varying degree of T lymphocyte deficiency (thymectomy, sham thymectomy, administration of antilymphocytic serum, B mice). The evidence obtained shows that the "lymphoid peak" recorded in the bone marrow during stress is likely to be formed at the expense of T and B lymphocyte migration from the peripheral lymphoid organs. The data have been also obtained, indicating that T lymphocytes migrating to the bone marrow during the first 6-9 hours after the exposure to stress may participate in granulocytopoiesis activation.     

Heterologous antiserum to rat lymphohemopoietic precursor cells.

Lymphohemopoietic precursor cells in rat bone marrow are members of a subset of lymphocyte-like cells that bears the bone marrow lymphocyte antigen (BMLA) and that lacks antigens present on peripheral B and T cells. This was demonstrated by two experimental approaches. In the first, bone marrow cells with the potential to form hemopoietic colonies in spleen (CFU-S), to repopulate lumphoid tissues and blood, and to rescue lethally irradiated recipients were enriched approximately 10-fold by a fractionation procedure designed to isolate a "null" population of bone marro lymphocytes. In the second approach, the lymphohemopoietic precursor cell activity in bone marrow was completely abrogated by opsonization with rabbit antiserum (ALSBM) raised against this "null" population of bone marrow cells. Precursor cell activity was not affected by treatment with antiserum to T and B cells. Quantitative cross-absorption studies showed that the antigen detected by ALSBM on lymphohemopoietic precursor cells had the same cellular distribution as did the previously described bone marrow lymphocyte antigen. It is likely that this antigen is present both on pluripotent stem cells and on committed progenitors of the myelocytic, erythrocytic and lymphocytic series.     

Detection of heterogenicity of the lymphoid populations by measuring the diameter of the cells and cell nuclei]

Some parameters of distribution according to the lymphoid cell and its nuclei size in the peripheral blood, bone marrow, thymus and spleen of healthy rats were studied. A comparative assay revealed population homogeneity for the thymus and bone marrow lymphocytes as well as their mean diameter differences. Mixing of these cell types markedly changed the distribution parameters of newly formed population, as shown on the model of the bone marrow lymphoid reaction caused by migration of thymic lymphocytes after 5-fluorouracil use. Preliminary thymectomy excluded migration and homogeneity of the bone marrow lymphocyte size remained unchanged.     

Two color light scattering identifies physical differences between lymphocyte subpopulations

Forward angle light scattering of two different wavelengths by cells in a flow cytometer was used to investigate physical differences between lymphocytes of different lineage, functional subclass and developmental stage. Correlation of the ultraviolet (UV: 351 nm and 364 nm) and 488 nm light scattering signals produced by lymphoid cells demonstrated that the two signals were not equivalent and that they placed different emphasis on the physical parameters characterizing lymphocytes. Both small T and B lymphocytes from peripheral lymphoid tissues and mitogenically activated large T and B lymphocyte blasts were discriminated by both wavelengths. Differences between the Lyt-2 negative and Lyt-2 positive T lymphocyte subsets were also apparent. Two color light scattering could also discriminate between immature thymocytes and mature peripheral T cells and between small bone marrow cells and mature peripheral B cells. In bone marrow an increase in UV light scattering coincided with the appearance of cell surface immunoglobulin on small cells. These data establish that two color light scattering is a sensitive probe for distinguishing cells of apparently similar morphology and that it can be used to study the physical changes that occur during lymphoid cell differentiation.     

B-Lymphocyte differentiation in lethally irradiated and reconstituted mice. II. Recovery of humoral immune responsiveness.

The recovery of humoral immune responsiveness was studied in lethally irradiated, fetal liver-reconstituted mice. By means of both membrane fluorescence and antibody formation to sheep red blood cells (SRBC) as a functional assay, the rate of recovery of the compartments of B and T lymphocytes was determined in various lymphoid organs. The recovery of the immunoglobulin-positive (B) cell compartment after irradiation and reconstitution started in the spleen. This organ was also found to be the first in which the recovery of the B-cell population was completed. The interval between the recovery of the B-cell population in the spleen and that in the other organs tested was found to increase when the irradiated mice were reconstituted with spleen colony cells instead of fetal liver cells. This proved to be caused by the number and nature of the reconstituting hemopoietic stem cells. The immunoglobulin-positive (B) cells were found to appear before SRBC-reactive B cells could be demonstrated in spleen, lymph nodes, and Peyer's patches. The appearance of T lymphocytes in the various lymphoid organs required even more time. By means of cell transfer experiments, a sequential appearance of the precursors of anti-SRBC IgM-, IgG-, and IgA-plaque-forming cells could be demonstrated in spleen, bone marrow, lymph nodes, and Peyer's patches.     

Monoclonal rat anti-mouse brain antibody detects Abelson murine leukemia virus target cells in mouse bone marrow

We report the characterization of a monoclonal antibody which detects a surface antigen expressed by the bone marrow target cell of A-MuLV. Treatment of bone marrow cells with this antibody and complement results in >95% loss of the A-MuLV-derived in vitro transformed foci. The surface antigen detected by this antibody is also expressed on A-MuLV-transformed lymphoid cell lines, thymocytes, and some peripheral lymphocytes. This antigen is not expressed, however, by the pluripotent hematopoietic stem cell defined by the spleen colony-forming assay. We present evidence that the antigen detected is neither a virally encoded product, nor exclusively associated with the BALB/c genome.     

Quantitative investigation of stem cell candidates in the bone marrow of growing guinea pigs.

Possible candidates for hemic stem cells were studied in a quantitative investigation of the bone marrow of growing guinea pigs. The stem cell candidates were classified as lymphoid cells of types I-IV, roughly corresponding to small lymphocytes, large lymphocytes, micromyeloblasts and myeloblasts of other authors. The cellular content of the bone marrow of guinea pigs of three different ages was determined. All types of nucleated cells per femur were found to increase with age. When the cellular content was related to the body weight, a peak number of lymphoid cells was found in the intermediate age group with the exception of the lymphoid cells of type III, which reached its peak in the oldest animals. This cell type has been suggested by Dicke et al. as the probable stem cell responsible for colony formation.     

Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.     

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.     

Two subpopulations of stem cells for T cell lineage

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.     

Characterization of the Growth Factor Activity of Amniotic Fluid on Cells from Hematopoietic and Lymphoid Organs of Different Life Stages

We investigated the proliferation-promoting effects of murine amniotic fluid (MAF) on in vitro cultured cells originally obtained from murine hematopoietic and lymphoid organs at different life stages. MAF promoted proliferation of the fetal liver cells (FLC), newborn spleen cells and adult bone marrow cells. The proliferation-promoting activity of MAF was extended to liver cells and spleen cells from mice younger than 2 weeks old. MAF did not, however, promote the proliferation of newborn or adult thymocytes, or of spleen cells, liver cells or peritoneal cells from 2-week-old or older mice. Rather, it partially inhibited the proliferation of spleen cells, thymocytes and peritoneal cells from 1-year-old mice. These results suggest that MAF contains growth factors for hematopoietic stem cells but not for either mature or immature T lymphocytes. Supporting this view, the MAF activity was partially neutralized by a polyclonal anti-mouse stem cell factor (SCF) antibody. Moreover, the immunoblotting of MAF against anti-mouse SCF antibody revealed a band at 30–32 kDa corresponding to the previously reported SCF. Interestingly, MAF was able to maintain FLC and adult bone marrow cells alive in culture for a relatively long time (2 weeks). The MAF activity was further shown to be partially and cell type-dependently antagonized by TNF-α and TGF-β. These results provided evidence that MAF contains potentially multiple growth factors preferentially affecting the early stage of hematopoiesis, one of which is SCF.