Effect of stimulating antibody formation under the influence of bone marrow cells in vivo]

Injection of intact bone marrow cells to mice at the peak of the secondary immune response results in a 2.4-fold increase of the number of antibody-forming cells in the regional lymph node. Preliminary injection of bone marrow cells to donors of the immune lymph node cells decreases the stimulation effect of antibody formation when the lymph node cells are subsequently cultivated with the intact bone marrow cells. The data obtained demonstrate the cell interaction at the level of mature antibody producers in vivo.     

Replacement of the helper function of T cells by an RNA-containing antigen-specific lysis factor]

The lymph node viable cells suspension of immunized mice was centrifugated. The supernatant was chromatographed in Sephadex G-200, and fractions were deproteinized. The deproteinized third fraction (Mol wt 30000) stimulated specifically the plaque-forming cells of intact mice immunized by SRBC. It restored the capacity to antibody production in the lethally irradiated intact mice protected by the syngeneic bone marrow. The activity of this fraction disappeared following treatment with RNA-ase, but not with DNA-ase or trypsin. The first and the second deproteinized fractions of the supernatant inhibited non-specifically the viable lymph node cells of the immunized animals in the intact mice immunized with SRBC.     

Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

Autoaggressive immunocompetent cells in the bodies of mice at remote periods following irradiation]

Lethally irradiated DBA/l mice or (C57Bl X DBA/l1 F1 hybrid mice were injected with therapeutically effective doses of isologous bone marrow cells; simultaneously syngeneic lymph node cells from either intact (control) animals or mice survived after sublethal irradiation were transplanted. In control the viability of the recipients was not affected by the presence of lymphoid cells in the mixed transplant. In contrast, the beneficial action of the bone marrow cells was abolished (killing-effect) by the lymphoid cells from mice sacrificed 6 to 12 months after the irradiation (600--700 r). The manifestation of the killing-effect depended on the number of the transplanted lymphoid cells and on the dose of the bone marrow cells in the transplant. The killing-effect was not revealed when the lymphoid cells were obtained from the donors on the 30th day after irradiation. The results suggest the autosensitization of the organism at the late postirradiation periods.     

Formation of an IgM and an IgG immune response depending on the redistribution of T- and B-subpopulations under the action of serotonin]

The syngeneic transfer of spleen cells or spleen and lymph node cells from donors with an elevated serotonin level stimulated, in comparison with the control animals, immune response in the recipients subjected to sublethal irradiation, which was manifested by an increase in the number of plaque-forming and rosette-forming cells. After the combined transfer of spleen cells and bone marrow cells from similar animals a decrease in the number of plaque-forming and rosette-forming cells was observed, while after the transfer of spleen and thymus cells the intensity of immune response remained unchanged. Serotonin was supposed to induce the redistribution of T and B cells in the non-immunized animals, so that suppressor cells migrated from the spleen and the lymph nodes to the bone marrow.     

Nature of the target cells in bone marrow B-suppressor action studied on a simplified model of suppression activity registration

The addition of bone marrow cells to spleen cells and lymph node cells stimulates by mitogents, but not to fibroblast-like cells, leads to a significant reduction of DNA synthesis in mixed cultures in vitro. The suppression effects appears only in two days and the suppressor cell activity is the stronger, the intensive is the target cell proliferation. It is shown that intact bone marrow cells can suppress the lipopolysaccharide-activated bone marrow cell proliferation in vitro. A conclusion may be draw that cells of the lymphoid system serve as target cells for the bone marrow suppressor cells, and the role of these lymphoid system cells is to control immunogenesis processes by suppressing the target cell proliferation activity in the bone marrow.     

Cryopreservation of hemopoietic tissue aimed at reducing immunological reactivity

A method has been developed of decreasing immunologic activity of lymphocytes from spleen, lymph nodes, and bone marrow (freshly collected or frozen) with a high percentage of intact stem cells. In experiments in vivo on lethally irradiated mice, it was demonstrated that during combined transplantation to the recipients of preserved bone marrow from two genetically different donors, a rapid decrease or absence of the effect of inactivation of hemopoietic stem cells under the influence of allogeneic lymphocytes was observed in the mixed graft. When it is necessary to transplant large quantities of bone marrow from several genetically different donors, the use of cryopreserved bone marrow is preferable to freshly drawn marrow due to the higher proliferative activity and the decreased risk in the development of immunological reactions.     

Correction of a T-immunodeficit in mice by bone marrow transplantation from donors treated with hydrocortisone]

Adult CBA mice were exposed to thymectomy, lethal irradiation, and protection by syngeneic bone marrow transplantation. In some experiments syngeneic bone marrow of donors, treated with hydrocortisone in a dose of 125 mg/kg for 3 days was used. The bone marrow of these donors contained cells with the Q-marker. Thymectomized and lethally irradiated animals subjected to the transplantation of syngeneic bone marrow from hydrocortisone-treated donors rejected the skin allotransplants, and the lymph node cells of these mice suppressed the endogenous colony-formation in the sublethally-irradiated hybrids (CBA X C57Bl/6) F1.     

Failure of ovalbumin associated with allogeneic spleen ceels to stimulate proliferation of lymph node cells of immune mice.

Ovalbumin-pulsed spleen cells were found to stimulate thymidine uptake of lymph node cells of syngeneic mice immunized with ovalbumin in complete Freund's adjuvant after treatment of spleen cells with Mitomycin C but not after heating the spleen cells at 56degrees for 30 min. Ovalbumin-pulsed spleen cells of allogeneic mice failed to stimulate the immune lymph node cells more than unpulsed cells, although a net increase in the thymidine uptake above the allogeneic stimulation was observed when free ovalbumin was added to the mixed culture. To eliminate the high background of the mixed lymphocyte reaction, F1 mice were made chimeric with bone marrow of one of the parental strains. Using lymph node cells of the immunized chimeras, the stimulation by pulsed spleen cells was much greater when antigen was presented on cells of the parental strain used for bone marrow injection than when presented on cells of the other parental strain.     

Suppression by bone marrow cells of the level of specific IgE antibodies in the blood of mice and decrease in the production of IGE by bone marrow cells

Changes in the level of the specific IgE-antibodies to ovalbumin under the influence of syngeneic cells of a bone marrow were studied. The IgE-response was induced by ovalbumin in mice (CBA X C57Bl/6)F1. The bone marrow cells suspensions (20-30 X 10(6) cells per mouse) from syngeneic donors was inoculated simultaneously with the immunization. It was found that bone marrow cells suppressed both the level of IgE-antibodies in experimental mice serum and the production of IgE by the bone marrow cells of the recipient. The ability to suppress IgE-response remained when erythrocytes, monocytes and T-lymphocytes were removed from inoculated suspensions. The bone marrow cells taken from the mice immunized with ovalbumin, at the stage of a decreasing IgE-response, provided more pronounced suppression, than bone marrow cells taken from intact animals.     

Studies of the mouse Ly-6 alloantigen system

Three monoclonal antibodies were produced by fusing mouse myeloma cell line NS-1 with spleen cells from C3H/An mice hyperimmunized with B6-H-2^k spleen cells. These antibodies recognized an alloantigen displaying a similar strain distribution pattern to the Ly-6.2 and Ala-1.2 alloantigens. Analysis of C×B and B×H recombinant inbred mice revealed close linkage of genes controlling Ly-m6 and Ly-6. The monoclonal antibodies lysed 70 percent of cells in lymph nodes and 60 percent in spleen in direct cytotoxicity assays, but did not lyse significant numbers of cells of thymus and bone marrow. Separated T and B cells were reactive with the antibodies, but T cells were more sensitive to the antibody and complement than B cells. Virtually all cells in cultures of cells activated in the mixed lymphocyte reaction or by Concanavalin A were reactive with the monoclonal antibodies. Direct plaque-forming cells were completely eliminated by the monoclonal antibody and complement. By absorption tests, cells from all organs tested so far (thymus, lymph node, spleen, bone marrow, brain, kidney and liver) were shown to express the Ly-m6 determinant. Tumor cell lines with T, B or stem cell characteristics were reactive with the monoclonal antibody by direct cytotoxicity and absorption assays.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

Differential response of B cells in the lymph node and the spleen to bacterial lipopolysaccharide

In vivo polyclonal activation of B cells in the lymph nodes and the spleens of mice injected with bacterial lipopolysaccharide (LPS) was compared. The peak of anti-trinitrophenylated sheep red blood cells plaque-forming cell (PFC) response in the lymph node was reached 6-8 days after the injection of LPS while that in the spleen was reached at 2 days. The maximal increase in the total number of Ig-producing cells in the lymph node also occurred at the later stage. These differences in time courses of polyclonal activation of B cells between the lymph node and the spleen were not due to the absence of B cells in the lymph node, migration of PFC from the spleen to the lymph node, or qualitative differences of B cells. This phenomenon was dependent on the environmental difference between the lymph node and the spleen, because B cells from the lymph node could respond to LPS rapidly in the spleen. Further, the polyclonal activation of B cells was accelerated in the lymph nodes of mice receiving prior injection of LPS. In in vitro cultures of lymph node cells of those mice, a significant amount of interleukin-1 could be detected by stimulation of LPS. It was possible that the delayed activation of B cells in the lymph node was due to the time lag necessary for construction of the environmental condition suitable for activation of B cells, whereas in the spleen this condition can be provided without delay.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Effect of tumor immunity on the distribution of labeled lymphoid cells in tumor-challenged mice

The distribution of ⁵¹Cr-labeled lymphoid cells from normal mice and mice immunized against a tumor were compared after intravenous inoculation of the labeled cells into normal syngeneic recipients. Spleen cell preparations from immune donors contained increased percentages of spleen and bone marrow-seeking cells, thus suggesting expansion of these cell populations when immunity to a tumor exists. Homing of labeled normal cells in tumor cell-injected normal animals was somewhat different from that seen in tumor cell-inoculated mice that were immunized against the tumor. In the latter case, accumulations of lymph node and spleen cells in recipient lymph nodes and bone marrow were consistently lower. In contrast, lymphoid cells from animals immunized against the tumor were found to accumulate in virtually the same percentages in lymphoid organs of normal and immune recipients. The behavior of lymphoid cell populations from thymus or bone marrow that consist mainly of precursor cells was unaffected by presence of malignancy and/or tumor immunity.     

Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells

Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body-irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells.     

Thymus dependency of cells involved in transfer of delayed hypersensitivity to listeria monocytogenes in mice

Delayed-type hypersensitivity (DH) to Listeria antigens was induced in inbred C3Hf/Umc mice by intravenous injection of a sublethal dose of viable Listeria monocytogenes. Bone marrow, spleen, and lymph node cells from the immune mice were capable of passive transfer of DH to syngeneic neonatally thymectomized or lethally (900 R) irradiated recipients. Immune thymus cells as well as immune serum were ineffective in transferring DH to irradiated animals. In vitro treatment with antitheta isoantibody (anti-θ) and complement abolished the capacity of spleen and bone marrow cells from immune donors to transfer DH to irradiated hosts, indicating the thymus dependency of this cell population. The results with bone marrow indicate the existence of a small, but biologically significant, thymus-dependent population in this tissue.     

Characteristics of the reactions to N-alloantigens of lymphoid cells from hydrocortisone-stimulated and adrenalectomized mice]

The capacity of the spleen, bone marrow and thymus cells from CBA mice (intact, adrenalectomized, and those treated with single or repeated hydrocortisone injections) to induce the lymph node type of "graft-versus-host" reaction (GVHR) in (CBA X C57BL) F1 hybrid recipients was evaluated. Two days after 2.5 mg hydrocortisone injection the capacity of the spleen and bone marrow cells to induce GVHR increased while that of the thymus cells remained unchanged. Seven and particularly 15 days after hydrocortisone injection the spleen cells became less active. Two days following repeated daily hormone injections in a dose of 0.25 mg within 18 days the thymocyte activity in GVHR increased, while that of the spleen and bone marrow cells did not change.     

Formation in agar of fibroblast-like colonies by cells from the mouse pleural cavity and other sources

Colonies of elongated fibroblast-like cells (stellate colonies) developed in agar cultures of mouse pleural cavity cells mixed with whole blood. Cultures of pleural cells alone developed only abortive clusters of round cells. The frequency of colony-forming cells in the pleural cavity was highest in neonatal mice (200/10⁵ cells) and fell progressively with aging. Stellate colony-forming cells were not in cell cycle but were radiosensitive. In adult mice, only occasional colony-forming cells were detected in peritoneal cavity, thymic, spleen, lymph node or bone marrow cell populations. Stellate colony formation was not stimulated by the granulopoietic regulator, colony stimulating factor. The active component in whole blood required for stellate colony formation was present in plasma but not serum or washed red or white cells.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.