Suppressor activity of spleen cells in drug-induced immunologic tolerance

Tolerance to sheep's red blood cells (SRBC) was induced in (CBA×C57BL/6)F₁ mice by a single intraperitoneal injection of 6×10⁹ SRBC followed by injection of 100–200 mg/kg cyclophosphamide 44–46 h later. Spleen cells of tolerant mice, obtained at various times (12–26 days) after induction of tolerance, when injected into intact syngeneic recipients, did not depress their immune response to SRBC. Unlike intact mice, tolerant mice were unable to produce suppressor cells after a single immunization with SRBC. Only if three additional injections of large doses (6×10⁹) of SRBC were given to the tolerant mice did their spleen cells acquire the ability to inhibit the immune response on injection into normal mice. It is postulated that the absence of suppressor cells on induction of immunologic tolerance by means of cyclophosphamide is due to clonal elimination. Suppressor cells may arise in tolerant animals under the influence of intensive antigenic stimulation, leading to deepening of the state of tolerance as a result of additional injections of SRBC.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 558–560, November, 1978.     

Effect of antiserum against isologous aggregated immunoglobulins on delayed-type hypersensitivity reaction in mice immunized with sheep red blood cells

Injection of mouse antiserum against isologous aggregated immunoglobulins (MAAS) into mice previously receiving 10⁵ sheep red blood cells (SRBC) did not affect the intensity of the delayed-type hypersensitivity (HDT) reaction when tested at the peak of sensitization (4th day), but led to a marked increase in the intensity of the reaction in the later stages (6th day). MAAS completely abolished suppression of HDT observed after sensitization with 5·10⁷ SRBC. Transfer experiments showed that under the influence of MAAS the number of suppressor cells of HDT in the spleen of the sensitized mice was reduced. MAAS had no effect on proliferation of antibody-forming cells or on the intensity of hemagglutinin production, but reduced by 70% the number of rosette-forming cells (RFC) detectable in the spleen at the peak of the primary immune response. The results may be evidence that RFC take part in the suppression of HDT.Laboratory of Immunochemistry, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 11, pp. 578–580, November, 1979.     

Immunodepressive action of cyclophosphamide on different strains of mice

The immunodepressive effect of cyclophosphamide (CP) was studied on mice of three strains (BALB/c, CBA, DBA/2) immunized with sheep's red cells (RBC). When the optimal immunizing dose of antigen was used (5×10⁸ RBC) the strongest immunodepression was observed in the DBA/2 mice, but when a large dose of RBC was used (6.2×10⁹) the strongest effect was observed in DBA/2 and CBA mice. The action of CP was shown to depend on the dose of antigen injected: In BALB/c mice the decrease in the number of antibody-forming cells was the same with both doses of RBC, in the DBA/2 mice an increase in the dose of antigen led to weakening of immunodepression, but in CBA mice immunodepression was intensified (provided that sufficiently large doses of CP were used). Determination of the rate of oxidative hydroxylation of CP by the mouse liver microsomes showed it to be comparatively low in DBA/2 and CBA mice and much higher in BALB/c mice. It is suggested that the differences in the immunodepressive action of CP thus revealed could be due to differences in the sensitivity of the target cells and (or) differences in its metabolism in mice of different strains.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. N. Zhakov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 438–440, April, 1977.     

Suppression of delayed type hypersensitivity in mice receiving massive doses of xenogeneic red blood cells

Injection of 6×10⁹ sheep's red blood cells into mice led to suppression of hypersensitivity of delayed type (HDT) in situ and caused activation of spleen cells suppressing sensitization of the recipients. Preliminary thymectomy on the donors and treatment of the cell suspensions with anti-T-globulin abolished the suppressor effect. Preliminary injection of small doses of cyclophosphamide potentiated both the response of antibody formation and the formation of HDT. With an increase in the dose of cyclophosphamide antibody formation was depressed and the HDT response further intensified. The results suggest that suppression of HDT is due to short-living, intensively proliferating cells of thymus origin and also, perhaps, B-cells.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 12, pp. 693–695, December, 1979.     

Mechanisms of hyporeactivity in mice after injection of lysed erythrocyte antigen

A state of specific hyporeactivity to sheep's red cells (SRBC) was induced in mice by injection of hemolyzed SRBC. Blocking serum factor in these mice was shown not to be identical in the character of its action with antired-cell antibodies, but to be probably an antigen-antibody complex. After combined injection of hemolyzed SRBC and cyclophosphamide (CP) into mice production of blocking serum factor was suppressed but the reactivity of the mice to SRBC was considerably reduced. It is suggested that in this case inactivation of the immunocompetent cells took place through the combined action of CP and SRBC antigen in a nonimmunogenic form.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. P. Bochkov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 253–256, March, 1979.     

The parental resistance phenomenon and its genetic regulation

Lymphocytes of (CBA×M523)F₁ or (A×M523)F₁ mice, if transplanted into CBA or A recipients irradiated in a dose of 1000 rad, react to test antigens (sheep's red cells,Salmonella typhi Vi-antigen) by the formation of only 1/100–1/1000 of the number of antibody-forming cells formed by syngeneic recipients. An intermediate result was observed after transplantation of the same cells into irradiated M523 recipients. Conversely, lymphocytes of (A×CBA)F₁, (CBA×C57BL/6)F₁, or (A×A.CA)F₁ mice gave an equal immune response in syngeneic recipients and in CBA or A recipients. The ability of M523 lymphocytes or their hybrids to give an immune response to sheep's red cells did not differ from the immuno-reactivity of lymphocytes of other lines either in situ or in a syngeneic adoptive system. Hematopoietic stem cells from (CBA×M523)F₁ mice formed only 40–50% of the number of colonies in the CBA spleen as in the spleen of syngeneic recipients. It is concluded that the M523 mutation interferes with the proliferation and differentiation of hematopoietic cells and lymphocytes in nonsyngeneic irradiated recipients.N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. Research Laboratory of Experimental Biological Models, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 247–250, March, 1979.     

Effect of regeneration of the spleen and bone marrow on number of hematopoietic colonies in the mouse spleen

Two thirds of the spleen (group 1) or the bone marrow from the right tibia (group 2) was removed from sexually mature male CBA mice. On the eighth day after lethal irradiation and injection of 1·10⁶ nucleated cells from the intact spleen the number of hematopoietic splenic colonies was counted. A significant increase in the number of colonies was observed in the animals of both experimental groups compared with the control intact mice. the authors suggest that this increase may have been caused both by the local effect of the regenerating splenic stroma and by a certain stimulating factor secreted by the regenerating hematopoietic tissue.Laboratory of Growth and Development, Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. Department of Histology, Biological Faculty, Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 11, pp. 1375–1376, November, 1976.     

A study of the lymph-node type of graft versus host reaction in mice

A local form of the graph versus host reaction (GVHR) was induced in adult (CBA×C57BL)F₁ hybrid mice by subcutaneous injection of semiallogeneic spleen, thymus, or bone marrow cells from CBA mice into the right hind footpad. The criteria of activity of the GVHR were an increase in the number of blast forms in the region of popliteal lymph node and in its weight 7 days after transplantation of cells. After transplantation of 5×10⁶ and 20×10⁶ spleen cells the absolute weight of the regional lymph node was increased by 3–5 times and was significantly higher than in the control (injection of living syngeneic or fragmented semiallogeneic cells from the same source). By contrast with the control, in the experimental animals the effect clearly depended directly on the dose of transplanted cells. Enlargement of the lymph nodes was accompanied by the regular appearance of blast forms in them. Thymus and bone marrow cells had a much weaker action than spleen cells.Department of General Pathology, Institute of Clinical and Experimental Medicine, Academy of Medical Sciences of the USSR Siberian Division, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 338–339, March, 1976.     

Resistance of the suppressor function of T cells to agents with antiproliferative activity

Injection of cyclophosphamide (CP) in a dose of 50–400 mg/kg into mice immunized with sheep's red blood cells (SRBC) does not significantly reduce the ability of the spleen cells of these mice to suppress the primary immune response when transplanted into intact syngeneic recipients. Irradiation of the donors of immune spleen cells (ISC) in a dose of 900 R or treatment of the ISC in vitro with mitomycin C did not affect their suppressor activity. The supernatant (SN) obtained after ultracentrifugation of sonicated ISC inhibited the primary immune response of intact mice. It is concluded that the suppressor effect of ISC is due to a factor produced by the T cells; active proliferation of these cells is not essential for the realization of its action.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 9, pp. 327–330, September, 1977.     

Factors determining long-term chimerism of lymphoid tissue in cyclophosphamide treated mice

The conditions of origin of long-term chimerism were investigated in adult CBA mice receiving cyclophosphamide and F₁(CBA×C57BL/6) spleen cells. Essential conditions are a high dose of cells (more than 50×10⁶), a dose of cyclophosphamide of not less than 200 mg/kg, and short intervals (3–6 h) between their injections. The results are interpreted from the standpoint of the hypothesis that cyclophosphamide can induce reversible injuries in lymphocyte DNA; these injuries become fixed after contact between lymphocytes and antigen and they lead to death of the corresponding cell alone.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Eyulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 2, pp. 194–197, February, 1976.     

Effect of preliminary injection of allogeneic cells on transplantation immunity in mice receiving cyclophosphamide

Injection of 1×10⁸ C57BL/6 mouse spleen cells into CBA mice 1 day before treatment with cyclophosphamide (CP) was shown to promote survival of 2×10⁷ allogeneic or semiallogeneic cells injected later (3–6 h after CP). The criterion of survival was the ability of the donor cells to produce antibodies against sheep's red blood cells in recipients tolerant to this antigen. Injection of 1×10⁸ allogeneic cells 2 days before CP treatment had no protective effect. After intravenous immunization with allogeneic cells, killer cells began to appear in the recipient's spleen as early as on the 2nd day, and their number reached a maximum on the 5th day. The results suggest that CP eliminates the recipient's lymphocytes responding to transplantation antigens, but the killer cells already formed are resistant to the action of CP.Laboratory of Immunologic Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 192–194, February, 1978.     

Rate of development of immunological memory and characteristics of the secondary immune response in mice of strains with high and low reactivity

Immunological memory for sheep's red blood cells develops in mice of strains CBA and DBA/2 and (CBA×C57BL/6)F₁ hybrids 24 h after injection of a small dose of the antigen, but 48 h after injection in C57BL/6 mice. The level of the secondary immune response in CBA, C57BL/6 and F₁ hybrids is significantly higher than in DBA/2 mice. Maximal production of antibody-forming cells in the spleen of the CBA mice is observed after two injections of small doses of the antigen. By contrast to this, to obtain a marked immune response in the case of adoptive transfer of spleen cells of C57BL/6 mice a second injection of a large dose of antigen is required.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. H. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 10, pp. 454–457, October, 1977.     

Effect of mouse antiserum against isologous aggregated immunoglobulins on accumulation of rosette-forming and antibody-forming cells in mice immunized with sheep's red blood cells

Data are given on the effect of mouse antiserum against isologous aggregated immunoglobulins (MAAS) on the kinetics of rosette-forming (RFC) and antibody-forming cells (AFC) in mice immunized with sheep's red blood cells (SRBC). The effect of MAAS in the experimentsin vivo was assessed by injecting this serum for 5 days into CBA mice, the first injecting being combined with injection of 5.10⁷ SRBC. Injection of MAAS into mice immunized with SRBC was shown to cause a marked decrease in the number of RFC in the spleen on the 5th and 9th days after immunization. MAAS has no appreciable effect at these same times on proliferation of AFC producing IgM hemagglutinins. Meanwhile MAAS intensified proliferation of IgG-AFC during the period when the number of these cells of the spleen in the immunized mice was maximal. After adsorption of MAAS with immune complexes formed by mouse IgG antibodies this serum was shown to lose much of its ability to block RFCin vivo. It is postulated on the basis of these results that the property of MAAS of influencing the accumulation of RFC and AFC producing IgG hemagglutinins is due to a factor which reacts with the immune complex formed by mouse IgG antibodies. This factor may perhaps be antibodies against aggregated immunoglobulins of this class.Laboratory of Immunochemistry, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 557–560, May, 1978.     

Quantitative analysis of the immunogenic activity of SV40 virus and of tumor cells induced by this virus

A study of specific antitumor immunity created in Syrian hamsters by oncogenic virus SV40 and by tumor cells induced by the same virus showed that the level of specific resistance depends on the immunizing dose of virus and cells. Investigation of the resistance-inducing activity of a wild strain of SV40 virus showed that the minimal dose inducing resistance in hamsters was ten times higher than for the tsA-30 mutant of the virus. The minimal resistance-inducing dose of irradiated cells of a tumor induced by the same strain of SV40 virus was 9·10⁵ cells; a tenfold increase in the dose led to a significant increase in the level of specific antitumor immunity.Laboratory of Immunology of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 8, pp. 223–225, August, 1978.     

Kinetics of colony-forming ability of mouse bone marrow cells after administration of hydrocortisone

The dynamics of the colony-forming and migration capacity of polypotent hematopoietic stem cells in the bone marrow of (CBA×C57BL) F₁ mice was studied after injection of hydrocortisone. The relative number of hematopoietic stem cells in the bone marrow was higher than in the control on the 3rd day after hydrocortisone injection. This increase was maximal on the 5th day after the injection. On the 8th day the number of hematopoietic stem cells was down to normal again.Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR V. P. Kaznacheev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 9, pp. 1102–1104, September, 1976.     

Effect of levamisole on the cytotoxic activity of normal lymphocytes in tissue culture

Preliminary incubation of normal nonimmune splenic lymphocytes from C57BL/6j and BALB/c mice for 45 min at 37°C with levamisole (1·10^–4 to 1·10^–5 M) followed by washing potentiated the cytotoxic effect against allogeneic target cells (L cells). When lymphocytes and levamisole were added to the culture of target cells simultaneously no cytotoxic effec occurred.Laboratory of Clinical Immunology, Oncologic Scientific Center, Academy of Medical Sciences of the USSR. All-Union Center for the Study of Antirheumatic Agents, Institute of Rheumatism, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol 88, No. 11, pp. 581–582, November, 1979.     

Stromal fibroblasts of hematopoietic organs and antibody formation in culture

Stromal mechanocytes of thymic, bone-marrow, and splenic origin obtained from monolayer cultures at the 3rd–6th passage, if added to suspension cultures of rabbit spleen cells by the method of Mishell and Dutton, have a significant effect on the accumulation of antibody-forming cells (AFC) by the 4th day in culture. Their action clearly depends on dose. Stromal mechanocytes of bone marrow origin, in doses of 2.1×10³–6.25×10⁵, caused inhibition of AFC formation in culture. Stromal mechanocytes of thymic origin in doses of 2.75×10³–8×10⁵ caused an increase in the number of AFC, whereas mechanocytes of splenic origin in doses of 2.1×10³–1.3×10⁴ had no significant effect, and in doses of 8×10⁴–6.25×10⁵ inhibited AFC formation. Many of the living cells and AFC were concentrated in the fraction of nonadherent cells.Laboratory of Immunomorphology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 49–51, July, 1978.     

Specific suppression of the immune response in the adoptive transfer system

Injection of spleen cells (SC) of syngeneic animals immunized with large doses of sheep's red cells (SRBC) into intact mice led to marked specific suppression of the immune response of the recipients. The highest suppressive activity was shown by SC taken from donors on the 14th day after intraperitoneal injection of SRBC. The SC of intact animals and of mice receiving a preliminary injection of rat red cells did not affect the immune response of the intact recipients on immunization with SRBC. Treatment of the immune SC with anti-T serum (ATS) or anti-B globulin (ABG) and complement considerably reduced or completely abolished the suppressive activity. Injection of a mixture of two suspensions of immune SC, one treated with ATS and the other with ABG, into intact recipients did not lead to suppression of the immune response. It is postulated that the suppressor cells in this particular model are T lymphocytes, expressing common antigens or antigens cross-reacting with B cells.Laboratory of Immunogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 571–573, May, 1977.     

Kinetics of hematopoietic stem cells in the bone marrow of hepatectomized mice

Two thirds of the liver was removed from (CBA×C57BL/6j)F₁ female mice. On the 5th day after the operation a significant increase was observed in the number of endogenous colonies in the spleen of the partially hepatectomized animals. This increase was not connected with a change in the number of stem cells in the bone marrow, for partial hepatectomy at different times after the operation did not affect the number of colony-forming units in the bone marrow.Laboratory of Virology, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Kraevskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 218–219, February, 1978.     

Primary and secondary immune response in animals after spontaneous loss of tolerance

The primary and secondary immune response was studied in mice in which tolerance was induced by injection of sheep's erythrocytes and cyclophosphamide (CP). In the early stages (from 1 to 8 weeks) after induction the mice were immunized with sheep's erythrocytes either in a single dose of 5·10⁸ cells or in two doses each of 1·10⁶ cells. Both methods of immunization gave equal results in the control animals. In the experimental animals the process of formation and (or) realization of immunological memory was impaired to a greater degree and recovered more slowly than ability to give a primary response.Laboratory of the Molecular Bases of Immunogenesis, Institute of Experimental Biology, Academy of Sciences of the Armenian SSR. Laboratory of Immunological Tolerance, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 6, pp. 716–718, June, 1977.