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.     

Regulation of lymphocyte production in the bone marrow. I. Turnover of small lymphocytes in mice depleted of B lymphocytes by treatment with anti-IgM antibodies

To examine the concept that the genesis of lymphocytes in the bone marrow may be regulated by homeostatic feedback signals from peripheral B lymphocytes or their products, lymphocyte production was measured in mice selectively depleted of B lymphocytes by repeated administration of anti-IgM antibodies from birth. The turnover of small lymphocytes was quantitated radioautographically after DNA labeling by continuous infusion of 3H-thymidine. In the femoral marrow of anti-IgM-treated mice, the number of small lymphocytes was reduced and their turnover time was shorter than in control mice, presumably reflecting the premature elimination from the marrow of maturing cells about to express surface IgM. The absolute number of small lymphocytes being produced per femur in unit time, however, was identical in anti-IgM-treated and control mice. Lymphocyte production in the thymus was also unaffected by anti-IgM suppression whereas in the spleen the turnover of small lymphocytes was reduced due to the lack of young immigrant B lymphocytes from the bone marrow. The results demonstrate that the normal large-scale production of lymphocytes in mouse bone marrow is independent of the magnitude of the peripheral pool of B lymphocytes or the level of circulating immunoglobulins, suggesting the process is not subject to feedback control. Some implications for the genesis and diversity of primary B lymphocytes are discussed.     

Acquisition of an anti-idiotypic cytotoxic T lymphocyte repertoire in B cell-transferred or tetraparental bone marrow chimeric mice

In previous studies we showed that major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) specific for the cross-reactive idiotype (CRI) of MOPC-104E myeloma protein could only be induced in BALB/c or BAB-14 mice which have the ability to produce the CRI, but not in C.AL-20 or C.B-20 mice which have no ability to produce the CRI. The strong correlation between CRI-specific CTL responder strains and CRI producers supports the idea that the VH gene products are intrinsic primary antigenic stimuli for the generation of the anti-idiotypic CTL. To investigate the role of B lymphocytes in the selection of T lymphocyte repertoire, the purified B cells of CRI producer strains were repeatedly injected into anti-CRI CTL nonresponder neonatal mice. CRI-specific CTL activity was successfully induced in the CRI nonproducer mice only when they were exposed to CRI producer strain B lymphocytes from neonatal life. When the CTL nonresponder adult mice received CRI producer B lymphocytes, the nonresponder phenotype was not changed into the responder phenotype. Inducibility of CRI-specific CTL was also analyzed in tetraparental bone marrow chimeras. When CRI nonproducer bone marrow cells repopulated along with CRI producer bone marrow cells, the anti-CRI CTL of CRI nonproducer origin were generated. Adaptive differentiation of haplotype preference was also observed. When these observations are taken collectively, we see that the anti-idiotypic T lymphocyte repertoire is not a genetically determined one, but rather that the repertoire of T lymphocytes strongly depends on the postnatal selection process through the intrinsic idiotypic repertoire of B lymphocytes, i.e., internal images.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

Regulation of bone marrow lymphocyte production: III. Increased production of B and non-B lymphocytes after administering systemic antigens

To examine the influence of exogenous stimuli on the genesis of lymphocytes in mouse bone marrow, the production rate and subsets of marrow lymphocytes were examined after a systemic injection of sheep red blood cells (SRBC). Radioautographic analysis after either pulse labeling or infusion of [³H]thymidine revealed a pronounced increase in the number of newly formed small lymphocytes appearing in the marrow, maximal 4–5 days after SRBC injection and dose related. The resulting expansion of the marrow lymphocyte population included both immature B cells and null cells, as shown by cell surface and cytoplasmic markers. Similar stimulation of marrow lymphocyte production followed an injection of either bovine serum albumin or mineral oil. No comparable stimulation occurred in either the thymus or the spleen. The results demonstrate that antigens and nonspecific irritants can exert a central effect in the bone marrow, producing a surge in the production of both primary B and non-B lymphocytes. The possible role of external stimulants in determining the normal rate of bone marrow lymphocyte production is discussed.     

The xid mutation affects hemopoiesis in long term cultures of murine bone marrow

Cells of the humoral immune system are particularly affected by a mutation at the X chromosome linked immunodeficiency disease (xid)locus. Although B cells are made in normal numbers, they fail to become phenotypically and functionally diverse. Consequently, poor antibody responses are mounted to certain types of Ag. There have been some indications that other types of hemopoietic cells may be influenced by the mutation and development of the humoral immune system is unusually dependent on the presence of T lymphocytes. We now describe an analysis of the lympho-hemopoietic environment studied with long term bone marrow cultures. Contrary to expectations, cultures initiated with cells from homozygous female or hemizygous male mice with the mutant allele established more quickly than normal. The accelerated initial growth pattern was clearly linked to the xid mutation. Artificial mixtures of marrow exhibited intermediate growth kinetics. Experiments with H-2 congenic and T6 chromosome marked cells did not reveal an intrinsic dominance of growth in nonadherent xid cells. Similar results were obtained with culture conditions which favored production of myeloid or lymphoid cells. These findings would be consistent with subtle changes in the bone marrow microenvironment resulting from the xid mutation. The pedigree of the mouse strains had a significant influence on lymphopoiesis in long term bone marrow cultures. Lymphocytes of BALB/c origin dominated over CBA/H background cells in cultures established from mixtures of the two, but this did not correlate with any functional deficiency in CBA/H stromal cells. In fact, establishment of an adherent layer was a rate-limiting step in initiating long term cultures and this could be achieved with a low dose inoculum of CBA/H marrow. Even more dramatic effects were found in hemopoietic cells from doubly defective C3H.nu/nu-xid mice. The bone marrow of these athymic animals contained normal numbers of granulocyte/macrophage progenitors. However, lymphoid cultures could not be reproducibly established with their cells and myelopoiesis was never observed in vitro. The relatively simple conditions which pertain in culture make it possible to appreciate effects of mutations and pedigree on hemopoiesis which are unremarkable in intact animals.     

Germinal center B cells and antibody production in the bone marrow

In secondary antibody (Ab) responses, Ag processing and presentation occur in secondary lymphoid organs but most serum Ab is produced by cells in the bone marrow. Plasma cells in the bone marrow are derived from B cells activated by Ag in secondary lymphoid organs. We hypothesized that germinal center (GC) B cells, which acquire Ag from follicular dendritic cells in draining lymph nodes during the first few days of the secondary response, migrate to the bone marrow to terminally differentiate and produce specific Ab. To test this we looked for GC B cells in the thoracic duct lymph and in peripheral blood after secondary challenge using the peanut agglutininhi phenotype and blast cell morphology as markers for GC B cells. In addition, GC B cells were injected i.v. into naive recipients to determine if they would home to the bone marrow. Finally, to determine if the bone marrow environment supports maturation and Ab production by GC B cells, we cocultured GC B cells with bone marrow cells or bone marrow supernatants. The results indicate that blast cells bearing the GC B cell phenotype were present in both the thoracic duct and the peripheral blood 3 days after antigenic challenge. Day 3 peripheral blood cells secreted specific Ab, whereas cells isolated on day 0, 8, or 11 did not. Furthermore, in adoptive transfer experiments, only the day 3 GC B cells produced specific Ab and migrated to the bone marrow of naive mice. Finally, either bone marrow cells or factor(s) produced by bone marrow cells markedly enhanced Ab production by day 3 GC B cells. These data support the hypothesis that during the first few days after secondary challenge GC B cells seed the bone marrow and differentiate into plasma cells which produce the large quantities of Ab typical of secondary responses.     

Autologous or syngeneic mixed lymphocyte reaction in bone marrow and thymic chimera mice

Unfractionated peripheral blood mononuclear (UM) cells from adult donors of known serological status wtih respect to Epstein-Barr (EB) virus were exposed to four or more successive in vitro stimulations with irradiated cells of the autologous EB virus-transformed cell line at a responder: stimulator ratio of 4:1, and effector UM and T cells were prepared after each stimulation. Ten out of fourteen seropositive donors and all four seronegative donors thus tested showed at best moderate cell proliferation over two or three stimulations only and a cytotoxic response which became dominated by non-E-rosette-forming cells active against the K562 cell line but not against EB virus-transformed lymphoblastoid lines. Cocultures from three other seropositive donors gave stronger proliferative responses and yielded effector cells dominated by a polyclonal E-rosette-forming population cytotoxic to the autologous and to certain allogeneic (both HLA-related and -unrelated) EB virus-transformed cell lines as well as to some but not all EB virus genome-negative hemopoietic cell lines of the kind sensitive to natural killer cells. With one other seropositive donor, this same repeated stimulation induced a quite different type of cytotoxic response, selectively amplifying an effector T-cell population which appeared on the basis of target cell specificity and of sensitivity to monoclonal antibody blocking to be both EB virus-specific and HLA-A and B antigen restricted in its function.     

A new molecular genetic diagnosis of the Btk(xid) mice.

A new method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of a point mutation of the Bruton's tyrosine kinase (Btk) gene in CBA/N mice bearing an X-linked recessive immunodeficiency (xid). Since restriction site useful for RFLP analysis does not exist in the spontaneous mutant Btk(xid) locus, an artificial restriction site was introduced by PCR amplification with a modified primer. The five genotypes of the Btk locus (Btk(xid)/ Btk(xid), Btk(xid) /Btk+ and Btk+/Btk+ females and Btk(xid)/Btk(null) and Btk+/Btk(null) males) could be distinguished by three patterns clearly and easily. This PCR-RFLP analytic method will be useful as a tool in the production of congenic mice and mice with multiple immunodeficient genes.     

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.     

Physiology of B cells in mice with X-linked immunodeficiency. I. Size, migratory properties, and turnover of the B cell pool

In terms of certain immune functions and density of surface IgM, B cells from xid mice are often viewed as the equivalent of the immature (Lyb-5-) B cell subset of normal adult mice. In this paper we examine xid B cells with regard to certain physiologic functions, including homing to the lymphoid tissues, recirculation, and turnover. Xid mice were found to possess about one-third of the total number of B cells found in normal mice. This applied irrespective of whether one examined the spleen, lymph nodes, or outputs of B cells in thoracic duct lymph. In terms of migration to spleen, lymph nodes, and Peyer's patches, capacity to recirculate from blood to thoracic duct lymph, and turnover, xid B cells proved to be indistinguishable from normal spleen or thoracic duct B cells. Within these parameters, most xid B cells closely resemble the normal mature long-lived population of B cells residing in the recirculating pool of normal mice. Because xid B cells are functionally quite different from normal mature B cells, it seems reasonable to view xid B cells as an abnormal population not represented in normal mice.     

Infection with the human immunodeficiency virus (HIV) is associated with an in vivo increase in B lymphocyte activation and immaturity

The expression of phenotypic markers on B lymphocytes in patients with the acquired immune deficiency syndrome (AIDS), in human immunodeficiency virus (HIV) seropositive individuals, and in healthy seronegative donors was examined by two-color flow cytometry. Patients with AIDS and HIV-seropositive individuals showed an elevated percentage of B cells bearing an activation marker, the transferrin receptor, when compared with donors not infected with HIV. A decrease in the percentage of resting (Leu-8 positive) B cells was also seen in AIDS patients and HIV-seropositive individuals. An increased percentage of circulating, immature (CALLA-positive, CD10) B cells was seen in AIDS patients. These phenotypic changes were accompanied by an increased level of spontaneous IgG and IgM secretion, and increased cell size within the total B cell population and in some B cell subpopulations, in patients with AIDS and in HIV-seropositive people. These results demonstrate that phenotypic changes indicative of in vivo B cell activation and immaturity accompany the polyclonal production of Ig seen in HIV-infected individuals.