Development of Natural Killer Cell Activity and Genetic Resistance to Bone Marrow Transplantation with Age: Effect of Neonatal Thymectomy

The effect of neonatal thymectomy on the development of splenic and bone marrow natural cell-mediated cytotoxicity and on genetic resistance to bone marrow transplantation was examined in mice. Natural cytotoxicity was measured by a ⁵¹Cr release assay; the ability to engraft foreign bone marrow was assayed by the spleen colony method. The natural cytolytic response of spleen cells increased progressively from youth to early adulthood, whereas that of the bone marrow declined during the same age period. Neonatal thymectomy significantly elevated the natural killer cell response of young mice only (4 weeks, spleen; 6 weeks, bone marrow). In other experiments, neonatally thymectomized and sham-operated mice were lethally irradiated at 4 or 6 weeks of age and injected with 2.5, 5.0 or 10 million rat marrow cells. Six days later spleen colonies were markedly reduced in both 4- and 6-week-old neonatally thymectomized mice with all rat marrow cell doses tested. Neonatal thymectomy did not alter the percentage of erythroid versus other colonies at either 4 or 6 weeks. In both thymectomized and sham-operated mice the number of colonies increased with increases in marrow cell dose. The data are suggestive of a production and dissemination to the spleen of cells involved in the natural cytotoxic response from the bone marrow.     

Induction of immunological tolerance by chimeric thymus transplantation

The role of bone marrow derived cells (BMD) within the thymus during the induction of immunological tolerance was investigated using transplantation of chimeric thymuses. Chimeric thymuses were constructed by reconstituting lethally irradiated BALB/C recipients with T-cell depleted C3H bone marrow (BM) cells. Two to three months post bone marrow transplantation the thymuses of these animals, in which the epithelium was of BALB/C origin and BMD cells of C3H origin (verified by immunoperoxidase staining), were transplanted into thymectomized, lethally irradiated BALB/C recipients which were reconstituted with T-cell depleted syngeneic (BALB/C) bone marrow cells. Induction of specific tolerance to the BMD cells (C3H origin) present in the chimeric thymus could be demonstrated in MLR at 3 to 6 months post chimeric thymus transplantation. It is concluded that bone marrow derived cells are able to induce immunological tolerance within the thymus.     

Stimulation of humoral antibody formation by polyanions. 3. Restoration of the immune response to sheep red blood cells by polyanions in thymectomized and lethally irradiated mice protected with bone marrow cells

Adult thymectomized C57BL/6J mice were lethally irradiated and injected 4 h later with 10 × 10⁶ syngeneic bone marrow cells. Two weeks later animals were either inoculated with sheep red blood cells alone or treated with synthetic polyanions such as dextran sulfate or polyacrylic acid prior to inoculation with sheep red blood cells. On the fifth day after immunization the mice treated with polyanions showed a 19 S and 7 S plaque-forming cell response as well as significant hemolytic titers. Control mice showed no significant plaque-forming cell response or hemolytic titers. Polyanions seem to be capable of replacing thymus cell function.     

Homogeneous immunoglobulins in the serum of irradiated and bone marrow reconstituted mice: the role of thymus and spleen.

The influence of thymectomy and splenectomy on the frequency and class distribution of homogeneous immunoglobulins (H-Ig) in serum was studied in lethally irradiated (DBA/2 x C57Bl/Rij)F1 mice reconstituted with syngeneic bone marrow. During four follow-up periods in the first 9 months after transplantation, the sham-operated controls and splenectomized animals developed transient H-Ig in an average frequency of 14.2 and 15.7% respectively. There were no marked differences in the incidence of H-Ig within these two groups. In contrast, thymectomized mice and mice both thymectomized and splenectomized showed H-Ig in much higher frequencies (average percentages 31.6 and 36.5, respectively). The highest frequency of H-Ig was observed between 1.5 and 3.5 months after transplantation. H-Ig of the IgG1 and IgG2 subclasses were most frequent in all groups during the first 3.5 months. Later, H-Ig belonging to the IgM class also appeared in somewhat higher numbers. H-Ig of the IgA class was a very rare finding at any time. These results indicate that the presence of the thymus, but not necessarily of the spleen, is an important factor in the regulation of the immunoglobulin heterogeneity during the reconstitution of the immune system in lethally irradiated and bone marrow reconstituted mice.     

The development of NK cell activity in thymectomized bone marrow chimaeras.

Most of the natural killer (NK) cells, recently derived from the bone marrow (14 days after injection of bone marrow cells into lethally irradiated mice), express the Thy-1 antigen; after some period of time (by day 28) a normal proportion (50%) of Thy-1+ NK cells is found. This study demonstrates that the shift of these Thy-1+ NK cells to Thy-1- NK cells is influenced by the thymus: NK cells developing in the total absence of the thymus--bone marrow (bm) cells from adult thymectomized (Tx) or nude (nu) mice injected into thymectomized recipients, (Tx) bm----(Tx) or nu bm----(Tx)--remain Thy-1+, while the presence of the thymus at some stage of the NK cell maturation--bone marrow cells injected into thymectomized recipients, bm----(Tx), or normal recipients reconstituted with bone marrow cells from thymectomized or nude mice, (Tx) bm----or nu bm----normal recipients--is sufficient for the development of normal ratio of Thy-1+ and Thy-1- NK cells.     

Acceleration of Immune Reconstitution after Bone Marrow Transplantation in Mice by Bone Marrow Stromal

To observe potential effect of the engineered bone marrow stromal cell line QXMSC1 secreting IL-6 (QXMSCIL-6) on accelerating immnune reconstitution in syngeneic bone marrow transplantation in mice, QXMSC1 was transfected with the eukaryocytic expression vector pcDNAIL-6, which contained hIL-6 cDNA by liposome-mediated gene transfecting technique. G418-resistance clone was selected by limiting dilution. The highest secreting clone was selected by ELISA assay and used in animal experiments. The recipient mice (BALB/c) were lethally irradiated and cotransplanted syngeneic bone marrow (10^7/mice) and the QXMSCIIL-6 (5×10^5/mice). Lymphocyte proliferation induced by ConA and LPS, helper T lymphocyte precursor (HTLp), cytotoxic T lymphocyte precursor (CTLp), plaque-forming cell (PFC), delayed type hypersensitivity (DTH) were examined 30, 60 days in post transplantation respectively. The results showed that lymphocytes proliferation to ConA and LPS, HTLp, CTLp increased, DTH and PFC were improved by cografted stromal cells QXMSCIIL-6 on 30, 60 days after BMT. These results demonstrated that the bone marrow stromal cell line QXMSC1 IL-6 transfected with IL-6 (QXMSC11L-6) accelerated immnune reconstitution in syngeneic bone marrow transplantation.     

In vivo and in vitro responses to tumor-associated antigens: apparent absence of T cell participation

The tumor-associated antigens of the murine tumors B16, SAD2 and C3HBA readily induced antibody production in vivo, but produced little transplantation immunity. These tumors grew better in normal syngeneic hosts than they did in lethally irradiated or thymectomized lethally irradiated, marrow reconstituted hosts, where they grew equally well. When blood or spleen cells were reacted with anti-H-2 sera directed against the responder cells or with anti-Θ C3H sera and complement and then cultured with irradiated syngeneic tumor cells or extracts of these tumors, the responses to the tumor-associated antigens and to two thymus-independent antigens were not impaired; however, the same treatment greatly reduced the responses to allogeneic tumor or spleen cells and to two thymus-dependent antigens. The observations suggested that the tumor-associated antigens under study primarily stimulated B rather than T cells.     

Further studies of thymus-bone marrow cell synergism in cutaneous manifestations of delayed hypersensitivity to methylated human serum albumin: The effect of cortisone acetate

The contribution of syngeneic bone marrow and thymus cell populations obtained from normal and cortisone-treated donor mice for reconstitution of delayed hypersensitivity to methylated human serum albumin (MHSA) was studied in lethally irradiated recipients employing a limiting dilution assay. Normal thymus cells contained at least one antigen-reactive cell (or cell unit) per 3.7 × 10⁷ cells, while cortisone-resistant cell populations contained one cell per 6.7 × 10⁵ cells. Thus, cells immunocompetent to MHSA are not destroyed by steroid. Bone marrow cell populations contribute effector cells, and as few as 2 × 10⁶ cells are capable of fully restoring delayed response when combined with optimum numbers of thymus cells. Large numbers of thymus cells alone will restore immune responsiveness, indicating that such populations do contain some effector cells, either as contaminating blood borne cells, or as glandular cells derived possibly from sinusoids. Similarly, bone marrow cells in larger numbers restore responsiveness, implying contamination with thymus-derived cells.     

An impairment of antibody production in adoptively restored, lethally irradiated thymectomized mice

Lethally irradiated, bone marrow protected, normal and thymectomized adult mice were given 140–250 × 10⁶ syngeneic spleen and lymph node cells from normal or sensitized donors. These mice were tested at intervals following irradiation for their ability to reject allogeneic and xenogeneic skin grafts and to produce haemagglutinins. The results show that adoptively restored, thymectomized mice are capable of responding normally to skin homografts of varying antigenic disparity and of producing haemagglutinins to xenogeneic antigens. However, the majority of the mice which received lymphoid cells from normal donors were unable to produce haemagglutinating antibody in response to allogeneic antigens. Both 19S and 7S antibody were produced by the thymectomized mice.     

Platelet-Derived Growth Factor Requirement for Cultured Descendants of Rabbit Stromal Bone Marrow Precursors

Addition of irradiated bone marrow feeder from guinea pigs and rabbits to cultures of passed rabbit bone marrow fibroblasts considerably decreases the efficiency of colony-formation and the total amount of fibroblasts in cultures. The presence of irradiated rabbit blood platelets sharply increases these parameters. The addition of irradiated bone marrow feeder to rabbit bone marrow fibroblasts cultured under conditions promoting cell differentiation also decreases the total content of fibroblasts in cultures. Our results suggest that cultured descendants of rabbit bone marrow stromal precursors preserve both the sensitivity to inhibitory factors produced by bone marrow cells coexisting with stromal precursors in cultures (typical of stromal precursors of these animals) and high sensitivity to growth-stimulating platelet-derived factors (typical of stromal precursors from rabbits and other animal species).__________Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 113–116, 2005     

Bone marrow origin of complement-receptor lymphocytes

Substantial regeneration of complement-receptor lymphocytes (CRL) took place in lymph nodes of adult thymectomized whole-body X-irradiated mice reconstituted with semi-allogeneic (F₁) bone marrow. In the presence of complement, alloantisera directed against the transplanted bone marrow killed more than 95% of the lymphoid cells of such chimeras. Moreover, lymph node cell suspensions and lymphoid tissue sections of neonatally thymectomized or adult thymectomized, irradiated and marrow-grafted mice contained an increased proportion of CRL. The results concur to establish proof that CRL are of extrathymic, bone marrow origin.     

In rat bone marrow Thy-1 antigen is present on cells with membrane immunoglobulin and on precursors of peripheral B lymphocytes.

Rat bone marrow cells carrying Thy-1 antigen were studied morphologically, and tested for their independence of the thymus and their relationship to the B lymphocyte lineage. Using a fluorescence-activated cell sorter to separate Thy-1⁺ and Thy-1^- fractions, it has been confirmed that up to 50 % of all nucleated bone marrow cells are Thy-1⁺, most of which have the morphology of small lymphocytes. Thy-1^-cells were mainly neutrophils and erythroid. Thy-1⁺ cells were found also in the marrow of B rats (rats thymectomized as adults, irradiated and reconstituted with syngeneic bone marrow from thymectomized donors drained of recirculating lymphocytes), though at a lower frequency (roughly half) than of normal rats. In both normal and B rats about 1/4 of the Thy-1⁺ cells also bore lymphocyte surface immunoglobulin (sIg), and these doubly labeled cells accounted for the majority (～ 2/3) of marrow cells carrying large amounts of sIg. Therefore, unlike mice, Thy-1 is not a marker of thymus-dependent lymphocytes in rats. The B precursor activity of marrow fractions was measured in a long-term re-constitution assay counting sIg⁺ cells in the thoracic duct of lethally irradiated recipients. Virtually all the precursors were in the Thy-1⁺ or sIg^- fractions, and were barely detectable among Thy-1^- or sIg⁺ cells. Thus, in the rat peripheral B lymphocytes descend from precursors bearing Thy-1 antigen but lacking sIg.     

MOP3, a component of the molecular clock, regulates the development of B cells

Differentiation and proliferation of haematopoietic progenitor cells occur in intimate contact with the bone marrow microenvironment which is composed of stromal cells and extracellular matrix proteins. MOP3 (also known as brain and muscle Arnt-like protein-1, BMAL1), a master regulator of circadian rhythm, plays important roles in the regulation of cell differentiation and general physical functions. In the present studies, MOP3-deficient mice had significantly reduced levels of B cells in the peripheral blood, spleen and bone marrow compared MOP3(+/-) or MOP3(+/+) littermates. Flow cytometry analysis showed the levels of pre-B cells in bone marrow of MOP3(-/-) mice were similar as that in control mice. Adoptive transfer of MOP3(-/-) bone marrow cells (BMC) to lethally irradiated BALB/c Rag2(-/-) recipients, normal T and B cell development was observed, whereas Adoptive transfer of BALB/c BMC to lethally irradiated MOP3(-/-) recipients, B-cell development was significantly impaired. These results presented herein with MOP3-deficient mice reveal the involvement of MOP3 in the development of B cells, but not other immune cells. The effect of MOP3 on the differentiation of pre-B cells to mature B cells might be mediated by the bone marrow microenvironment. This study also showed a connection between a master regular of circadian rhythm with B-cell development in mice.     

Stimulation of DNA synthesis in mouse lymphoid cells by polyanions in vitro. I. Target cells and possible mode of action

The effect of dextran sulfate on [³H]dThd incorporation in lymphoid cells was investigated. The polyanion activated DNA synthesis in spleen and bone marrow cells of normal mice. The highest rate of activation was detected in spleen cells of athymic (nude) mice; the ratio of [³H]dThd incorporation was higher in TxBM spleen cells of thymectomized, lethally irradiated and bone marrow protected mice than in spleen cells of normal donors. Thymus cells of normal mice could not be stimulated, but a slight response was obtained in cortisone-resistant thymus cells. A synergetic effect was found in normal thymus cells using a combination of dextran sulfate and phytohemagglutinin (PHA). In contrast, lipopolysaccharide and PHA showed no synergetic effect. The possible mode of action of polyanions as de-repressors of DNA synthesis is discussed.     

Response of lymphocytes to plant lectins: I. A thymic-dependent lymphoid population responsive to Pokeweed mitogen

The response of lymph node, bone marrow, splenic and thymic lymphocytes to phytohaemagglutinin and Pokeweed mitogen has been studied. Depletion of theta positive or recirculating lymphocytes abolished the response to phytohaemagglutinin but did not impair the response to Pokeweed mitogen. Lymphocytes from thymectomized or athymic `nude' mice respond poorly or not at all to phytohaemagglutinin or Pokeweed mitogen.

We conclude that the normal response to Pokeweed mitogen depends upon an intact thymus gland. This despite the fact that one population of Pokeweed mitogen-responsive cells are theta-negative bone marrow-derived cells. This conclusion is based upon: (1) the equal reactivity of theta-positive and theta-negative lymphocytes to Pokeweed mitogen as shown by the normal response to Pokeweed mitogen of anti-theta serum treated lymphoid populations and (2) the impaired response of lymphocytes from nude mice and of lymphocytes from thymectomized, irradiated and bone marrow reconstituted mice to Pokeweed mitogen.

     

Thymosin restoration of cellular immunity to Blastomyces dermatitidis in T-cell-depleted mice.

The immunopotentiating properties of thymosin in thymectomized, lethally irradiated, bone marrow-reconstituted mice (ThyXBM) were characterized, using footpad sensitivity to Blastomyces dermatitidis. Normal mice were shown to exhibit increasing delayed-type hypersensitivity responses to killed yeast cells of B. dermatitidis after injections of the organism on days 0 and 7, as measured by footpad swelling tests. The footpad response of normal thymosin-treated mice was similar to that of normal, non-thymosin-treated mice. ThyXBM mice were unable to elicit a footpad response when similarly injected and footpad tested with B. dermatitidis. Thymosin-treated ThyXBM mice responded to footpad testing at a level that was 62% greater than the response seen in non-thymosin-treated ThyXBM mice. This peak response occurred on day 12. The results indicated that thymosin was unable to enhance immune responses of normal intact mice but could restore immunocompetence in a T-cell-depleted host, as measured by footpad sensitivity to B. dermatitidis.     

Immunotherapy of experimental cancer by intralesional injection of emulsified nonliving mycobacteria: comparison of Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis.

Mycobacterium bovis (BCG), Mycobacterium phlei, and Mycobacterium smegmatis were each tested in emulsified form for their potency to cause regression of transplants of a syngeneic murine fibrosarcoma and of a syngeneic guinea pig hepatoma. On a weight basis, M. phlei and M. smegmatis were as effective as BCG in causing tumor regression. M. phlei and M. smegmatis were comparable to BCG in provoking delayed cutaneous hypersensitivity reactions in guinea pigs sensitized to M. phlei or M. smegmatis. In BCG-sensitized guinea pigs, M. phlei and M. smegmatis provoked weaker delayed cutaneous hypersensitivity reactions than did BCG. Purified protein derivative of M. tuberculosis was more active in eliciting delayed cutaneous hypersensitivity in BCG-sensitized guinea pigs than in animals sensitized with M. phlei or M. smegmatis.     

Pathogenesis of Lassa virus infection in guinea pigs.

A rodent model for human Lassa fever was developed which uses inbred (strain 13) and outbred (Hartley) guinea pigs. Strain 13 guinea pigs were uniformly susceptible to lethal infection by 2 or more PFU of Lassa virus strain Josiah. In contrast, no more than 30% of the Hartley guinea pigs died regardless of the virus dose. In lethally infected strain 13 guinea pigs, peak titers of virus (10(7) to 10(8) PFU) occurred in the spleen and lymph nodes at 8 to 9 days, in the salivary glands at 11 days, and in the lung at 14 to 16 days. Virus reached low titers (10(4) PFU) in the plasma and brain and intermediate titers in the liver, adrenal glands, kidney, pancreas, and heart. In moribund animals, the most consistent and severe histological lesion as an interstitial pneumonia. In contrast, the brain was only minimally involved. The immune response of lethally infected strain 13 guinea pigs, as measured by the indirect fluorescent antibody test, was detectable within 10 days of infection and was similar in timing and intensity to the fluorescent antibody test response of both lethally infected and surviving outbred animals. In contrast to the fluorescent antibody response, neutralizing antibody developed late in convalescence and was thus detected only in surviving outbred guinea pigs. The availability of a rodent model for human Lassa fever in uniformly susceptible strain 13 guinea pigs should facilitate detailed pathophysiological studies and efficacy testing of antiviral drugs, candidate vaccines, and immunotherapy regimens to develop control methods for this life-threatening disease in humans.     

Pathogenesis of attenuated Junin virus in the guinea pig model

The purpose of this work was to elucidate the pathogenesis of attenuated Junin virus (JV) strains in the guinea pig model. Three groups of guinea pigs were infected by the IM route with 10(3) PFU of the XJC13 and XJO-attenuated strains or with the XJ pathogenic strain of JV, respectively. Viremia was studied at 3, 5, 7, 9, 12, and 14 days postinfection (pi) (a) in serum samples of all animals and in washed cells from XJC13-infected guinea pigs by conventional techniques and (b) in whole blood samples from XJC13 and XJO animals by coculture with Vero cells. Virus spread was studied at 14 days pi in brain, spleen, lymph nodes, and bone marrow by parallel suckling mouse inoculation or organ homogenates and coculture of cell suspensions with Vero cells. By coculture techniques of whole blood, an otherwise undetectable viremia was demonstrated for both attenuated strains throughout the observation period. In contrast, XJ viremia was easily detected by direct techniques, as has already been shown. Attenuated virus was also shown to reach brain and bone marrow when coculture methods were employed. But titers were always markedly lower than those of the pathogenic strain. The sustained viremia demonstrated in guinea pigs infected with either attenuated strain explains the mode of viral dissemination and accounts for viral rescue and antigen detection from some organs. These results suggest that attenuated strains do not differ greatly in their invasive capacity in guinea pigs, but later on viral replication is impaired. Therefore, these findings reveal potential risks and should be noted when developing human vaccines.