Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

Detection of Mls-antigens in various mouse strains using a new method

Mice of strains CBA and BALB/c, when injected with lymphocytes from theH-2-compatible Mls-antigen-incompatible strains C3H and DBA/2, respectively, develop a reduced lymphocyte reactivity against cells of the injected strains as measured in the mixed lymphocyte culture (MLC). The mechanism of the development of a depression of the MLC response against Mlsantigens is unknown. In this investigation we have tested the MLC response of lymphocytes from CBA mice preinjected with C3H lymphocytes against cells from 12 different strains. It was observed that the response decreased against cells from strains C3H, AKR, and A/Sn. Infusion of CBA mice with AKR lymphocytes decreased their MLC response against the same three strains. In contrast, infusion of CBA mice with A/Sn lymphocytes reduced their MLC responses against strains C3H, DBA/2, and the congenic strains A/Sn, A.SW, A.CA, and A.BY. BALB/c mice which were infused with DBA/2 lymphocytes developed reduced responses against DBA/2, C3H, and AKR. On the basis of these results we propose that mice of our strains C3H and AKR possess a common Mls-antigen which is strongly stimulatory, and that DBA/2 mice possess a second Mls-antigen which is also strongly stimulatory. The congenic strains A/Sn, A.SW, A.CA, and A.BY, which have differentH-2 complexes, possess a third Mls-antigen which is less stimulatory. The Mls-antigens of the strains listed above seem to exhibit extensive immunological crossreactivity.     

Properties of reticulum cell sarcomas in SJL/J mice. III. Promotion of tumor growth in irradiated mice by normal lymphoid cells.

Experiments were performed to elucidate the mechanisms by which lymphocytes obtained from an M-antigen-incompatible strain reduce the specific mixed lymphocyte culture (MLC) response of lymphoid cell populations after injection into allogeneic recipients. Mice of strain CBA were injected with spleen cells from hybrids of the H-2-compatible, M-antigen-incompatible strain C3H. Normal C3H × CBA spleen cells increased the MLC reactivity of the host's lymphocytes during the first 1–3 days, and thereafter the response against C3H was drastically reduced. Mitomycin-treated or antibody-coated C3H × CBA cells rather enhanced the MLC responsiveness. Roughly similar results were obtained by injecting untreated H-2-incompatible C3H hybrid lymphocytes. Lymph node or spleen cell populations from CBA mice, injected with C3H × CBA cells up to 2 weeks earlier, were found to depress the MLC reactivity against C3H when transferred to new CBA hosts. The results indicate that injected cells had survived for 2 weeks in the host. On the other hand, H-2-incompatible C3H hybrid cells could not be detected even at day 3 after injection into CBA mice. The results also indicate that C3H hybrid lymphocytes have to be functionally intact and able to survive in the host for a relatively long period of time to be able to reduce the specific MLC response of the host's lymphocytes.     

Immunocompetent lymphoi- cells from pregnant mice studied in the graft-versus-host reaction]

The capacity of lymphoid cells taken from C57BL/6 mice gravid from the CBA males (the second trimester) to induce the graft-versus-host reaction in the hybrids (CBA X C57B/6) F1 was reduced as compared with the cells of the virgin donors and syngeneic gravid mice. This was expressed by the prolonged survival of the experimental recipients and reduced inhibition of endogenous colony formation in the spleen of the sublethally irradiated (500 r) hybrids. At the end of gravidity this capacity was restored, in some instances even exceeding control figures.     

Lethality in LPS-induced endotoxemia in C3H/HeCr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin

C3H/HeCr mice are more susceptible to infection compared with other strains. Lactoferrin (LF), a protein involved in innate defense, was shown to protect mice against lethal endotoxemia. In this investigation we attempt to explain the cause of increased susceptibility of C3H/HeCr mice to LPS and lack of protective LF action in these mice. We found that C3H/HeCr mice produced up to 5-fold more serum TNFalpha and 66% higher IFNgamma levels in response to i.v. LPS injection than the control, CBA strain. 24 h pretreatment of C3H/HeCr mice with LF did not cause inhibition of the LPS-induced TNFalpha serum levels, whereas in CBA mice LF significantly decreased TNFalpha level. IL-6 serum levels, in turn, were lowered in C3H/HeCr mice but elevated in CBA mice. That differential regulation of cytokine production by LF in C3H/HeCr mice paralleled a decreased survival after lethal LPS injection - 10% vs. 60% in control, PBS treated mice. In addition, determination of colony forming units (CFU) in livers and spleens after administration of 10(8) Escherichia coli revealed that pretreatment of CBA mice with LF caused a marked reduction of CFU in these organs, whereas in C3H/HeCr mice the changes were insignificant. These results indicate that the altered TNFalpha/IL-6 ratio in C3H/HeCr mice, as compared to control CBA mice, as well as the increased IFNgamma level, may be responsible for the increased susceptibility to endotoxemia in that substrain. We also suggest that an association exists between the LF protective effect against endotoxic sequelae and the insult-induced systemic immune response.     

Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius

Dobson C., Brindley P. J. and Sitepu P. 1982. Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius. International Journal for Parasitology12: 567–572. Different strains of serum donor mice showed variations in innate immunity to primary infections with Nematospiroides dubius. Different levels of anti-N, dubius antibodies were detected in sera from these mouse strains; there was no correlation between antibody titre and numbers of worms recovered. Serum from donor wild and six laboratory strains of mice protected female Quackenbush (Q) recipients against N. dubius infections; donor mouse strain influenced the degree of protection conferred and donor serum antibody titre related to the degree of stunting of worm growth in recipient mice. Five laboratory strains of mice developed different levels of protective immunity following multiple experimental infections with N. dubius. Antibody titres in these mice were strongly correlated with the percentage protection observed after 1–4 infections: Q and CBA mice produced more anti-N. dubius antibody and were better protected than DBA/2, BALB/c and C3H mice. However BALB/c, C3H and CBA mice attained similar anti-N. dubius antibody titres after a single infection with N. dubius but serum from BALB/c gave better protection when transferred to female Q recipients than that from the other two strains. This suggested qualitative differences in the protective antibodies in sera between mouse strains. Five mouse strains were passively immunized with a uniform dose of serum from female Q donors: DBA/2 female recipients showed the least, BALB/c and C3H females were intermediate, and Q and CBA female mice attained the greatest level of passive protection against N. dubius. A close positive correlation existed between the degree of actively acquired and the level of passively acquired protection between the five strains of mice.     

Interaction of thymus lymphocytes with histoincompatible cells. IV. Mixed lymphocyte reactions of activated thymus lymphocytes

CS7BL-activated CBA T cells (T.TDL) were obtained by thoracic duct cannulation of (CBA × C57BL)F₁ mice 4 days after heavy irradiation and injection of CBA thymus cells. T.TDL behaved differently from the TDL of normal CBA mice in unidirectional mixed lymphocyte culture in a number of respects: (a) the response of T.TDL was directed specifically against C57BL antigens, whereas normal TDL responded to both C57BL and BALB/c antigens; (b) the response of T.TDL was rapid but transient compared to that of TDL; (c) whereas only approximately 3% of TDL synthesized DNA specifically in response to C57BL antigens, as many as 25% of C57BL-activated T.TDL responded to these antigens. Evidence is presented which suggests that the T.TDL have a very limited capacity to proliferate. Most of the cells which responded to antigen synthesized DNA without subsequently entering mitosis.     

Induction of tolerance against the Mls antigen in mice no need for Mls-bearing cells

Infusion of CBA mice with spleen cells from the H-2-compatible, but Mis antigen-incompatible, strain C3H leads to a specific reduction of the MLC reactivity of the host's lymphocytes. One explanation of the reduced reactivity could be that the specifically Mls antigen-responsive CBA T cells become exhausted by intense antigen stimulation or that the infused cells actively neutralize specifically responsive cells. In this investigation, we have shown that depletion of membrane Ig-positive cells from C3H × CBA spleen cell preparations strongly reduces their capacity to stimulate CBA lymphocytes in the MLC, indicating that the Mls antigen is expressed on B but not on T cells. However, such B-cell depleted cell preparations were fully capable of reducing the MLC response of CBA hosts. Cell preparations of spleen and lymph nodes exhibited high stimulatory and inhibitory effects. Thymic cells lacked both these characteristics, whereas bone marrow cells were weak stimulators but relatively strong inhibitors. The results support the proposal that the observed reduction of MLC reactivity is due to an active process of the injected cells. The cell type which is working as an inhibitor has not been clearly defined yet.     

In vitro spleen cell responsiveness to various analogs of MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice

N-Acetylmuramyl-l-alanyl-d-isoglutamine (MDP), a synthetic immunoadjuvant, was incubated with spleen cells of DBA/2 or Balb/c mice and optimal responses were obtained after 4 or 5 days of culture in a serum-free medium supplemented with 2-mercaptoethanol. In contrast, lymphocytes of (C57B1/6 × AKR)F₁ hybrids responded weakly under the same conditions. The results reported here show that like in the case of DBA/2 and Balb/c strains, spleen cells of Swiss mice and of inbred AKR and CBA mice could be stimulated in vitro whereas C57B1/6 and LPS-refractory C3H/He mice did not respond. Fourteen synthetic MDP analogs (eight known to be adjuvant active and six devoid of activity) were tested in DBA/2 high-responder mice. A good correlation was observed between in vitro stimulation and the presence or absence of adjuvant activity in vivo of these compounds.     

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.     

Heterogeneity of the effectors of natural killer activity in CBA mice

The natural cytotoxicity of Percoll fractions of CBA mice splenocytes was tested against cells of human erythroblastosis suspension culture K-562 and murine C3HA hepatoma solid culture MGXXIIa. The 1.076 g/ml dense fraction was shown to have the highest activity in 3H-uridine cytotoxic test against the cell targets. Immunosera prepared by the Golub method against CBA mouse brains and exhausted supplementary by syngeneic thymocytes decreased the natural cytotoxicity of CBA mouse splenocytes against MGXXIIa tumor cells and, on the contrary, increased it against K-562 tumor cells.     

Studies on antigen-induced arthritis in mice. III. Cell and serum transfer experiments.

Antigen-induced arthritis in mice occurs after immunization and subsequent intraarticular challenge with methylated bovine serum albumin (mBSA). In adoptive transfer experiments, susceptible C57BL mice and resistant CBA mice were compared in their capacity to express delayed-type hypersensitivity (DTH) by ear assay, and to express arthritis. The expression of DTH could be transferred incrementally by lymphoid cells in C57BL mice, but not in CBA mice. Both immune lymphoid cells and, to a much lesser extent, serum transferred the capacity to develop arthritis in C57BL mice. The reactivity of transferred cells was abolished by anti-Thy-1 but enhanced by enrichment for T cells with anti-immunoglobulin columns. If this model disease can be equated with human rheumatoid synovitis, the lesions in the human disease would be an expression of a T cell-dependent activity.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

A vaccine based on exosomes secreted by a dendritic cell line confers protection against T. gondii infection in syngeneic and allogeneic mice

Our results show that exosomes secreted by SRDC pulsed in vitro with Toxoplasma gondii-derived antigens (Exo-TAg) induced protective responses against infection with the parasite in both syngeneic and allogeneic mice. After oral infection, syngeneic CBA/J mice exhibited significantly fewer cysts in their brains and allogeneic C57BL/6 mice survived. This protection was associated with strong humoral responses in vivo in serum from both CBA/J and C57BL/6 mice, and with high levels of anti-TAg IgA antibodies in intestinal secretions from CBA/J mice alone. Furthermore, strong cellular responses in vivo were observed in both mouse models. Cellular proliferation was associated with cytokines production by spleen and mesenteric lymph node cells. The results presented here show that exosomes are nucleic acid free vesicles that are able to induce immune responses correlated with protection against parasitic infections in both syngeneic and allogeneic mice. They could constitute an efficient tool for use in vaccination and antitumor strategies based on exosomes.     

RECOVERY IN THE MOUSE SPLEEN AFTER SUBLETHAL IRRADIATION

1. CBA and C57B1 mice were exposed to LD_50/30 doses of γ-radiation and at intervals after irradiation the numbers of haemopoietic colony-forming units (CFU) and of antibody-producing cells in the spleen were determined. 2. CFU repression was observed after transfer of non-irradiated cells to hybrid recipients. However, this repression did not appear until 60 days after irradiation of the donor. 3. The recovery after irradiation of the capacity to inactivate CFUs in spleen cell mixtures was studied. The results suggested that cells which produce 19S antibody are different from those which are reactive against foreign cells.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

Biophysical characterization of peyer’s patch lymphocytes in the B-cell deficient CBA/N mice and in their F1 hybrids

CBA/N mice bear an X-linked immunodeficiency involving abnormalities of their B lymphocytes. We investigated the electrophoretic mobility (EPM) distribution and the electronic volume of the Peyer's patch (PP) cells of these mice in comparison with those of the immunologically normal CBA/J mice. The same studies were also carried out on PP cells from the CBA/N×DBA/2 (CND2) F1 hybrid males and females that are, respectively, B-cell defective and normal. In all types of mice, PP cells could be separated by free-flow electrophoresis into a low-mobility (LM) population corresponding mainly to B cells and high-mobility (HM) population containing mostly T cells. However, while in the control mice LM (B) cells accounted for around 60–67% of PP lymphocytes, this population did not exceed 40% in the defective animals. Furthermore, LM (B) cells, but not HM (T) cells, of CBA/N and CND2 male mice were found to display abnormal physical characteristics. Thus, they possessed a higher anodic EPM (two electrophoretic fractions faster) and a slightly larger model size (134 μm³) than normal adult B cells (122–124 μm³). Similar differences were observed with PP lymphocyte preparations enriched for B cells by nylon wool adherence. These data suggest that CBA/N mice and their defective hybrids lack a population of small B lymphocytes with low anodic EPM.     

Anti-β1 Integrin IgG Inhibits Pulmonary Macrometastasis and the Size of Micrometastases from a Murine Mammary Carcinoma

In the present report, we investigated the possible importance of β₁ integrins in the growth and metastasis of a murine mammary carcinoma, SP1, and a metastatic variant, SP1-3M in vivo. CBA/J female mice bearing SP1 tumor transplants were injected with anti-β₁ integrin IgG or control nonimmune IgG (200 μg per mouse; i.p.) every two days. Animals received anti-CD4 antibody (100 μg per mouse) at time zero to suppress immunity against rabbit IgG. Outgrowth of macroscopic metastases from SP1, but not from SP1-3M primary tumors, was markedly inhibited in animals receiving anti-β₁ integrin IgG but not nonimmune IgG. To assess the stage(s) in the metastatic cascade affected, we examined the number and diameter of micrometastatic nodules in treated and untreated groups. The diameter of micrometastases was significantly reduced in SP1-tumor-bearing mice treated with anti-β₁ integrin IgG compared to control IgG, although the number of nodules per cm² of lung sections examined remained unchanged. No change in the number or size of micrometastases in SP1-3M tumor-bearing mice was observed. No difference in the binding, or complement-mediated and antibody-dependent cell-mediated cytotoxicity of anti-β₁ integrin IgG with SP1 and SP1-3M cells was detected. The results suggest that under these conditions anti-β₁ integrin inhibits metastatic tumor growth in lung tissue, but has minimal effect on intravasation, adhesion to target organs and extravasation.     

Effect of prodigiosin and its combination with immunodepressants on the graft versus host reaction in mice

The local (lymph node) graft-versus-host reaction (GVHR) in F1 (CBA X C57BL/6) mice and the lethal GVHR in C57BL/6 mice were induced by transfer of lymph node cells of CBA mice with skin allotransplants from C57BL/6 mice. Prednisolone in combination with asathioprin (imuran) administered to CBA mice inhibited the GVHR. Prodigiosan used alone was not active, while in combination with immunodepressants it increased their inhibitory effect. Adhesive cells with a suppressive activity were detected in the spleen of mice treated with prodigiosan. Such cells were capable of suppressing the capacity of syngeneic lymphocytes for inducing the GVHR.     

Marked difference in sensitivity to gamma-ray-induced cataract between BALB/c and CBA-C3H strain mice]

Nineteen BALB/c, 14CBA/KI, 12C3H/HeJ and 15 (CBA/Kl x C3H/HeJ) F1 female mice were irradiated with 850 rad gamma-rays and transferred with 10(7) syngeneic bone marrow cells 24 hrs later. The occurrence of cataract was examined in these animals. All the BALB/c mice showed visible lens opacification in both eyes between 113 and 149 days after irradiation. All the animals were autopsied 6 months after irradiation and examined for opacification of their lenses. The proportion of opaque lenses was 100, 7.1, 16.7 and 0% in BALB/c, CBA/Kl, C3H/HeJ and (CBA/Kl x C3H/HeJ) F1 mice, respectively. The results indicate that BALB/c mice are much more sensitive to radiation-induced cataractogenesis than CBA and C3H mice.