Modulation of suppressor activity by thymosin

Modulation of suppressor cell activity by thymosin was evaluated in vivo in a murine tumor system, and in vitro on human lymphocytes. We showed that splenocytes from tumor bearing mice were able to enhance tumor growth in a syngeneic system. This enhancement was T dependent and disappeared by Thymosin treatment. A decrease of Tumor size associated with injection of bone marrow cells treated by thymosin was observed. In the human system we showed that ConA stimulated lymphocytes were able to suppress the response of normal lymphocytes to PHA, PWM and ConA, and in MLC. This effect was significantly blocked in presence of thymosin fraction 5.     

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.     

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.     

Failure of NZB spleen to respond to prethymic bone marrow suppressor cells.

Mouse bone marrow contains theta-negative lymphocytes that can suppress an in vitro plaque response by spleen cells primed in vivo with burro red blood cells (BRBC). These bone marrow cells are radiosensitive and can be induced with thymosin fraction 5 or alpha 1 thymic peptides to express the theta antigen. Enrichment for these suppressor pre-T lymphocytes can be achieved by a one-step density centrifugation, macrophage depletion, or a combination of both procedures. NZB mice, which spontaneously develop an autoimmune disorder, have a suppressor abnormality revealed by this assay system. Upon analysis, they have normal BM pre-T suppressor cells but their spleen cells are refractory to the BM suppressor signal. NZB BM suppressor cells inhibit the response by DBA/2 spleen cells, but DBA/2 BM suppressor cells do not inhibit NZB spleen. This resistance to suppression is a property of the B cell fraction recovered from NZB spleen.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

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.     

Reaction of human lymphocytes with aggregated IgG columns. Effect on antibody-dependent cytotoxicity and EAC rosette formation.

The origin and life span of long-lived small lymphocytes in the bone marrow of mice have been evaluated by the use of radioautography, scintillation counting, and anti-theta serum. Thymus-deprived BALB/C mice and nude mice had a smaller percentage of long-lived lymphocytes in bone marrow and in thoracic duct lymph than sham-operated or normal littermates. Furthermore, the long-lived lymphocytes in the marrow of nudes have more varied—but generally shorter—life spans than long-lived lymphocytes from mice having a thymus. In thoracic duct lymph of nude mice a more homogeneous long-lived population—according to life span—was found.It was concluded that both long-lived T cells and long-lived B cells are normal residents in the bone marrow. Furthermore, it was concluded that cells of variable life spans comprise the B lymphocyte population: short-lived cells with life spans of 3–5 days and long-lived lymphocytes with life spans of weeks to months.     

Pre-B cells: bone marrow persistence in anti-mu-suppressed mice, conversion to B lymphocytes, and recovery after destruction by cyclophosphamide.

Chronic treatment of mice from birth with anti-mu antibodies aborts development of B lymphocytes and plasma cells. In these studies we show that bone marrow from anti-mu-treated mice contains a population of cells with cytoplasmic IgM, but which lack detectable cell-surface IgM. These cells are analogous to pre-B cells, defined in ontogenetic studies as the immediate precursors of B lymphocytes. Pre-B cells from bone marrow of anti-mu treated mice retain their functional integrity, as evidenced by their ability to give rise to sIgM+, LPS-responsive lymphocytes in culture. We also show that cyclophosphamide treatment destroys pre-B cells and that recovery of pre-B cells in bone marrow precedes the regeneration of sIgM+ B lymphocytes. Generation of B lymphocytes in adult mice apparently occurs exclusively in the bone marrow because induction of extramedullary hemopoiesis in spleen was not accompanied by the appearance of pre-B cells in that organ.     

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.     

Scanning electron microscopy of homing and recirculating lymphocyte populations

The surface structure of T and B lymphocytes in vivo was investigated using scanning electron microscopy. For these studies the spleen and mesenteric lymph node of mice enriched for B lymphocytes (adult thymectomized, lethally irradiated, bone marrow reconstituted mice, B mice) and of mice enriched for T lymphocytes (adult, lethally irradiated, thymocyte transferred mice, T mice) were examined. Both types of lymphocytes demonstrated a smooth cell surface when they were situated in their respective microenvironment, whereas recirculating T and B cells exhibited numerous microvilli on the cell surface. In postcapillary venules, known to be the major sites of entry of lymphocytes in lymph nodes, lymphocytes were in contact with the endothelial wall by means of these microvilli. While passing the endothelial lining, lymphocytes withdrew their microvilli and appeared smooth upon arrival in the lymphatic stroma. It is suggested that microvilli on the surface of lymphocytes play a role in cellular recognition mechanisms.     

Changes in the populations of null, NK1.1+, and Thy1lo lymphocytes in the bone marrow of tumor-bearing mice: effect of indomethacin treatment.

Mouse bone marrow produces many "null" lymphocytes which lack B and T lineage markers (B220-Thy1-). A subset of these cells expresses the natural killer (NK) cell marker, NK1.1. In addition, some rapidly renewed bone marrow lymphocytes express low intensities of Thy1 (Thy1lo). In view of their possible implication in tumor-host interactions these various cell populations have now been examined in mice injected with either the nonmetastatic Ehrlich ascites (EA) tumor or the Lewis lung carcinoma (LLc), a highly metastatic solid tumor. In each case, the number of null lymphocytes, as defined by a lack of radioautographic labeling of either B220 glycoprotein or Thy1, increased markedly in both the bone marrow and spleen. Treatment with the prostaglandin inhibitor, indomethacin, enhanced the increase in null cells in the bone marrow and spleen of LLc-bearing mice. The number of null small lymphocytes expressing NK1.1, as detected by combined radioautographic and immunoperoxidase techniques, increased almost 30-fold in LLc-bearing mice. The number of Thy1lo small lymphocytes increased in parallel with null cells during EA tumor growth. The findings accord with the hypothesis that the null lymphocyte population produced in mouse bone marrow includes newly formed NK lineage cells which sequentially express NK1.1 and Thy1lo. The present work demonstrates that the populations of null, NK1.1+, and Thy1lo lymphocytes in mouse bone marrow expand rapidly during the early growth of transplanted tumors, the initial increase in null lymphocytes apparently being curtailed by prostaglandin production. The results suggest that the production of null lymphocytes in mouse bone marrow is responsive to tumor development, possibly providing cells to be involved in tumor-host interactions.     

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.     

Characterization of small lymphocytes in the bone marrow of mice after prolonged treatment with anti-IgM antibodies.

In mice treated with anti-IgM antibodies from birth, small lymphocytes in the bone marrow and spleen have been characterized by their incidence, surface markers, and turnover. Essentially complete elimination of IgM-bearing small lymphocytes followed injections of anti-IgM but this treatment did not deplete the IgM-negative small lymphocytes in the marrow. These IgM-negative cells also lacked other markers of B lymphocytes, such as receptors for Fc and complement; they failed to bind anti-mouse T-lymphocyte serum and they formed a rapidly renewing population within the marrow. This population may represent cells whose normal differentiation has been aborted by the anti-IgM antibodies or, alternatively, they may be “null” cells, distinct from B lymphocytes.     

Lymphohemopoiesis in culture is prevented by interaction with adherent bone marrow cells from mutant viable motheaten mice

Mice with the recessive "motheaten" (me) or "viable motheaten" (mev) mutations have severe immunologic disturbances and die at an early age. The function of hemopoietic progenitor cells and microenvironmental elements that regulate their growth and differentiation were studied in mev mice with two types of long term bone marrow cultures. Cells from bone marrow of homozygous defective mev/mev mice were non-productive under conditions that normally support replication of stem cells and production of neutrophil granulocytes. Similarly, in a different culture system, lymphocytes were produced from normal littermate, but not mev/mev bone marrow. Initial overgrowth of cells having macrophage-like characteristics occurred in both culture systems with marrow from defective mice. Co-cultures of normal and defective bone marrow cells were always non-productive. In contrast, supernatants of mev/mev bone marrow cultures did not have a detrimental effect on cultures of normal cells, implying that the suppression was cell-associated. Furthermore, there was no evidence for abnormal release of granulocyte or macrophage growth factors in mev bone marrow cultures. A small population of cells in mev/mev bone marrow cultures were morphologically similar to "stromal" cells that support lymphohemopoiesis. Certain culture strategies could be used to enrich for these. mev/mev stromal cells had affinity for normal lymphocytes; however, they did not support lymphocyte growth. The long term bone marrow cultures thus reveal an apparent imbalance in the regulatory mechanisms affected by these single gene mutations. This is manifested by preferential or aberrant growth of one type of adherent cell and a possible functional abnormality of stromal cells. mev mice could provide an ideal model for investigating cell-associated molecules that normally limit progenitor cell replication.     

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.     

Bone marrow-derived T lymphocytes responsible for allograft rejection

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

Development of C3 receptors on B lymphocytes derived from normal and memory marrow cells.

Differentiation of B lymphocytes was studied by adoptive transfer of bone marrow into irradiated mice which were then assayed for generation of splenic lymphocytes with complement receptors. These cells developed to normal numbers between 20 and 25 days following marrow cell transplant. Quantitative enhancement of CRL generation occurred when recipient mice were antigen stimulated prior to assay. CRL levels returned to those of normal adults sooner than those found in mice which were not antigen stimulated. A third form of CRL development occurred in mice that were reconstituted with bone marrow cells from antigen-primed donor mice. Here complement receptor-bearing cells appeared much earlier but increased in numbers at a slower, more constant rate to a maximum level on Day 25. Attempts to T-cell deplete recipient mice did not alter receptor development.     

20 alpha hydroxysteroid dehydrogenase (20 alpha SDH) activity in New Zealand mice T lymphocytes and bone marrow cells: effect of age, sex, and castration.

The activity of 20 alpha hydroxysteroid dehydrogenase (20 alpha SDH), a T lymphocyte-associated enzyme, was measured in fetal liver, thymus, spleen, and bone marrow cells from NZB, NZW, and (NZB X NZW)F1 (B/W) mice. There was an age-dependent increase of 20 alpha SDH activity in bone marrow cells, and a decrease in thymocytes and splenic T lymphocytes. Treatment with anti-theta and complement did not reduce the 20 alpha SDH activity of bone marrow and fetal liver cells, but reduced the activity of spleen cells. PHA stimulates both 20 alpha SDH activity and thymidine incorporation in splenic, bone marrow, and fetal liver lymphocytes. The results suggest that the enzyme in the bone marrow and fetal liver is located in pre-T lymphocytes. Enzymatic activity in bone marrow cells taken from female B/W mice (older than 7 months) was 40 to 20% lower than in male mice. Orchidectomy, but not ovariectomy, caused a significant decrease in thymocyte 20 alpha SDH activity. Orchidectomy depressed and ovariectomy enhanced 20 alpha SDH activity of bone marrow cells. The 20 alpha SDH activity of fetal liver cells from B/W mice was twice as high as in either parent strain. No 20 alpha SDH activity was found in fetal liver cells taken from BALB/C SJL or C57BL/6 mice. A model is proposed to explain the age- and sex-related changes in 20 alpha SDH activity of pre-T and T lymphocytes in healthy and pathologic conditions.     

Studies on the mitogen responses of germfree allogeneic chimeras. II. Maturation of two cell types and partial restoration of responsiveness of the short-term chimeras.

Germfree allogeneic bone marrow chimeras (ABMC) were produced by the i.v. injection of approximately 10(7) bone marrow cells from germfree DBA/2 mice into lethally irradiated germfree C3H mice. In the germfree state, the short-term ABMC showed no histologic signs of graft-vs-host reactions (GVHR), yet splenic lymphocytes were unable to respond to PHA, Con A, or SRBC. Attempts to remove responsiveness by the implantation of a DBA/2 thymus under the host kidney capsule also resulted in failure. However, when the donor thymus was enclosed in a cell-impermeable chamber to eliminate a GVH reaction, responsiveness to Con A was restored. The PHA and SRBC responses were unaffected by this treatment. Daily injections of thymosin caused both an increased Con A response and increased numbers of PFC, although the PHA response was again unaffected. Thus, soluble substances from thymic tissue can be used to overcome partially the histocompatibility barrier present in the ABMC that affects at least two different functional cell populations.     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.