Impairment of T-helper function by a Plasmodium berghei-derived immunosuppressive factor

Mice injected with an immunosuppressive factor (ISF) extracted from Plasmodium berghei-infected rat erythrocytes have a reduced antibody response to unrelated antigens. T-cells from ISF-treated mice failed to provide adequate help to naive, syngeneic B-cells in the primary IgM response in vitro to sheep red blood cells and to dinitrophenylated keyhole limpet hemocyanin. The same T-cells, however, were able to cooperate with memory B-cells in the secondary IgG response. No other cellular deficit was detected in ISF-treated mice; B-cells and macrophages behaved normally, and there was no detectable excess of suppressor cells. The T-cell impairment was not reflected in decreased production of interleukin 2, but was also shown by the diminished delayed type hypersensitivity reaction to sheep red blood cells of ISF-treated mice.     

Replacement of T-Cell Function by a T-Cell Product

WE have recently shown that the immune response to sheep red blood cells (SRBC) in vitro is T-cell dependent, that is, it can be abrogated by pretreatment of the spleen cells with anti-θ serum and complement¹. We further reported that reconstitution of the system can be achieved by, among other things, the addition of allogeneic thymocytes², while syngeneic thymocytes failed to function as helper cells. Positive allogeneic effects were only obtained if the added thymocytes could recognize the remaining B-cells as foreign. One of the explanations evoked for this allogeneic effect was the participation of a potentiating factor^2,3. It seemed possible that on the heavy antigenic stimulation provided by the histocompatibility antigens carried by the B-cells, added T-cells produce a soluble factor which stimulates the immune response of B-cells to unrelated antigens. We now want to report the existence of two distinct factors, one of which can completely replace T-cells.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Ca++-dependent immunosuppressive effect of ethylenediaminetetraacetic acid (EDTA)]

Subcutaneous injection of a sum total dose of 6.44.10(-5) M of ethylenediaminetetraacetic acid (EDTA), which binds divalent cations, inhibits the process of accumulation of antibody-forming cells in the spleen of mice (CBA X C57BL) F1 hybrids, immunizated with sheep red blood cells. In a dose of 5.2.10(-5) M CaCl2 eliminated the immunodepressive effect of EDTA almost completely. It was assumed that EDTA action was produced through the Ca++-controlled regulation mechanism of one of the following processes: the interaction between T- and B-lymphocytes; proliferation and differentiation of T- and B-cells in the course of the immune response to sheep red blood cells. A more complex mechanism of EDTA action on the above-mentioned processes is not excluded, however.     

A study of the ability of Bordetella pertussis toxin to induce the formation of B-suppressors

Mouse spleen cells not adhering to the plastic surface and B-cells isolated from them were treated with B. pertussis toxin in vitro, washed and injected into recipients (allogeneic, syngeneic, intact or lethally irradiated) whose immune response to sheep red blood cells was then evaluated by Jerne's method. Treatment with B. pertussis toxin was shown to induce the development of immunosuppressive activity in intact spleen cells and in B-cells, to abolish the activity of memory B-cells and to enhance the suppressor activity of autoimmune mice. Supernatants obtained after autoimmune mice. Supernatants obtained after the 18- to 24-hour cultivation of spleen cells, previously treated with B. pertussis toxin for 60 minutes, suppressed the reaction of blast transformation of spleen cells to Con A and lipopolysaccharide and induced the appearance of immunosuppressive activity in intact spleen cells. The suppressing effect of the cells studied in this investigation may be linked with the ability of B. pertussis cells to stimulate the synthesis of cAMP, prostaglandins E and/or suppressor factors.     

Interaction and migration of T- and B-lymphocytes in immune responses during carcinogenesis induced by methylcholanthrene]

The (CBA X C57BL) F1 mice were injected intramuscularly with methylcholantrene (MCA) in a dose of 0.3 mg, and their T- and B-cells ability to cooperate in the immune response against sheep red blood cells, and also migration of these cells from the thymus and the bone marrow to the spleen were studied. The MCA immunosuppressive action proved to be associated with the inhibition of migration and cooperation of T- and B-lymphocytes in the immune response. A conclusion was drawn that the immunosuppressive effect developing during the carcinogenesis was complex and it was realized at various stages of immunogenesis.     

Functional activity of splenic T- and B-cells during the process of tumor growth]

Functional activity of B- and T-cells was studied in mice during the growth of the inoculated tumours; it appeared that in the course of the tumour growth the capacity of B- and T-cells to the interaction in the immunization with sheep erythrocytes was disturbed. B-cells were affected earlier than T-cells.     

Effect of highly purified thymus factor on the cellular and humoral indices of immunity in thymectomized mice]

Effect of homogeneous polypeptide thymus factor of mol weight about 5000 (thymarin-III) on cellular and humoral immune responses of thymectomized adult CBA mice was studied. Thymectomy proved to greatly decrease the number of T-cells in the spleen. Accordingly, the capability of these mice to produce both IgM and IgG antibody-forming cells in response to the thymus-dependent antigen (sheep red blood cells) was significantly depressed. Subcutaneous injections of thymarin-III (1 microgram per g of body weight) for 7 days completely restored the T-cell spleen population and normalized the animals' immune response.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4⁺ and CD8⁺ T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4⁺ and CD8⁺ T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1 Gy of X-rays, CD8⁺ T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4⁺ and CD8⁺ T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells.

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4(+) and CD8(+) T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4(+) and CD8(+) T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1Gy of X-rays, CD8(+) T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4(+) and CD8(+) T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Resistance of the suppressor function of T-cells to agents possessing an antiproliferative action]

Administration of cyclophosphamide in a dose of 50 to 400 mg/kg to mice immunized with sheep red blood cells failed to decrease significantly the capacity of the splenic cells of these mice to suppress the primary immune response in transplantation to intact syngeneic recipients. Irradiation of the donors of immune splenic cells (ISC) in a dose of 900 r or treatment of ISC in vitro with mitomycin C failed to influence their suppressor activity. Supernatant obtained after the ultracentrifugation of ISC treated with ultrasound inhibited the primary immune response of intact mice. A conclusion was drawn that the suppressor effect of ISC was caused by the factor produced by T-cells. Active proliferation of these cells was not necessary for the realization of its action.     

B- and T-cell tolerance induction in young-adult and old mice.

Young-adult and old (CS7BL × C3H)F₁ mice were injected with either of two tolerogens followed by challenge with dinitrophenylated human γ globulin (DNP-HGG). The primary IgM and IgG responses of spleen cells to the DNP determinant were evaluated by a modified hemolytic plaque assay. Carrier-specific thymus-derived cell (T-cell) tolerance was induced in mice with deaggregated HGG. Hapten-specific bursal-derived cell (B-cell) tolerance was induced with DNP coupled to isogeneic mouse IgG. The dose of these two tolerogens was successively decreased by 10-fold decrements until a response similar to that of control mice was achieved. The minimum tolerizing dose (MTD) was then determined for young-adult and aged B-and T-cells. We found that the MTD for old B-cells was 10 times greater than that obtained with young B-cells for both the direct and indirect PFC responses. No difference in MTD was observed between young and old T-cells when assessed by the indirect response; the MTD for old T-cells was 10-fold greater than that obtained for young-adult T-cells when the direct response was evaluated. A double threshold of tolerance was found for T-cells of young-adult mice.     

Tolerance-like condition developing in mice under the effect of antigen of erythrocyte lysate]

Lysate of sheep red blood cells obtained by the treatment of these cells with distilled water and purified by ultracentrifugation in cold possessed a weak immunogenicity. Its administration to mice caused the state of hyporeactivity to sheep red blood cells (a reduction of the immune response level to 10-25% of control. The capacity of the mise spleen cells to respond by immune reaction to the red blood cells following adoptive transfer was not disturbed. At the early periods after the lysate administrations the mouse spleen cells possessed a weak supressive activity in case of their transfer to the intact animals. The blood serum of mice treated with the lysate possessed a blocking activity which disappeared after the serum absorption with sheep red blood cells. A conclusion was drawn that hyporeactivity originating in mice after the lysate administration was caused by the presence in the serum of antibodies inhibiting the immune response.     

Effect of the antineoplastic antibiotic bruneomycin on lymphocyte population ratio in mice

Data on the quantitative ratio of the populations of T- and B-cells in the lymphoid organs of mice at various periods after oral administration of bruneomycin are presented. It was shown that in contrast to the total number of T- and B-cells in the spleen and mesenteric lymph nodes amounting to the minimal values at the early periods (days 1-3) after the antibiotic injection, their relative content remained rather high. Complete recovery of the number of T- and B-lymphocytes in the above organs was observed only by the end of a month period. Study on the kinetics of the immune response to sheep red blood cells showed that reactivity of the antibody producers in the experiments with bruneomycin increased 1.5-2 fold as compared to the control.     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Patterns in the antigen-nonspecific modulation of the B-cell activity in mice under the influence of a purified staphylococcal anatoxin

Purified staphylococcal toxoid (PST) has been shown to be an antigen-nonspecific immunomodulator, capable of inducing changes in the immune response of B-cells to unrelated antigens, such as sheep red blood cells (SRBC), in a wide range of doses (from 15 to 0.15 binding units per mouse). The manifestation of the immunomodulating effect depends on the conditions of the experiment: the doses of PST and SRBC, the age of mice, the sequence of the injections of the antigens and the intervals between the injections. The simultaneous injection of PST and SRBC induces, as a rule, an increase in immune response to the test antigen, while their separate injection induces mainly immunosuppression.     

Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.     

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.     

Effect of an orally applied herbal immunomodulator on cytokine induction and antibody response in normal and immunosuppressed mice

The influence of the oral administration of a herbal immunomodulator, consisting of an aqueous-ethanolic extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix, on cytokine induction and antibody response against sheep red blood cells was investigated in mice. The treatment of the animals with the extract caused no enhancement of the cytokine titers in the serum. Spleen cells isolated from the treated mice, however, produced higher amounts of IL-2, IFNgamma and GM-CSF ex vivo in comparison to spleen cells isolated from control animals, especially after additional stimulation by lipopolysaccharides or concanavalin A. The application of the extract also triggered the production of IL-1 and TNFalpha by peritoneal macrophages ex vivo. The influence of the herbal extract on the antibody response was examined by the plaque forming cell assay. The administration of the extract caused a significant enhancement of the antibody response against sheep red blood cells, inducing an increase in the numbers of splenic plaque forming cells and the titers of specific antibodies in the sera of the treated animals. In mice, immunosuppressed by old age or additional treatment with hydrocortisone, the therapy with the extract resulted in a normalization of the antibody response against sheep red blood cells.     

6-(2,4-Dinitrophenyl)-mercaptopurine, a stronger immunosuppressive drug than 6-mercaptopurine to primary humoral T-cell dependent immune response in mice]

The suppressive effect of 6-(2,4-Dinitrophenyl)-mercatopurine (DNP-MP) and 6-mercaptopurine (MP) was investigated on the early primary immune response of mice against the T-cell dependent antigens DNP49-bovine gamma globuline (BGG), sheep red blood cells (SRBC) or FITC8-BCG and the T-cell independent DNP22-Ficoll. The number of IgM antibody forming cells (AbFC) to the hapten determinants and to the SRBCs per 10(6) spleen cells was determined. DNP-MP reduced the number of AbFCs after the immunisation with the T cell dependent antigens always stronger than the MP, independently of the antigen type by which the mice had been immunised. The Anti-DNP22-Ficoll immune response was suppressed equally by both immunosuppressive drugs. DNP-MP is not a specific immunosuppressive drug for the anti-DNP-B-lymphocytes. Helper T-cells and macrophages are discussed as target cells for the stronger unspecific action of DNP-MP.