Inhibition of the growth of a syngeneic weakly immunogenic tumor in mice resulting from exposures affecting T lymphocyte activity

Adult BALB/c mice were thymectomized, lethally irradiated and reconstituted with cells of syngeneic embryonic liver (B-mice). The growth of the syngeneic low-immunogenic tumor of spontaneous origin (Acatol) was strongly inhibited in B-mice as compared to that in intact recipients. The transplantation of the tumor to adult-thymectomized hosts 3 months after operation also resulted in marked retardation of tumor growth as compared to intact or sham-operated animals. The same effect was observed in mice preimmunized with spleen cells from tumor-bearers but not from intact donors. It is inferred that BALB/c mice possess strong non-specific factors of tumor resistance. However, they are actively suppressed by the mature immune system. Apparently, tumor cells, regardless of low immunogeneity are antigenic enough for the syngeneic host and induce a series of immune reactions, bringing about activation of T suppressors. It is assumed that attempts at immunizing a tumor host with autochthonous T suppressors might lead to a promising approach to cancer immunotherapy.     

Characteristics of the reaction of immunologically tolerant mice to the polyclonal stimulant salmozan

The nonspecific stimulant of the immune response salmozan (Sal) increases the number of PFC against SRBC in intact mice, B mice (thymectomized, irradiated and reconstituted with embryonic liver cells), and in mice pretreated with cyclophosphamide (CY). This effect was decreased in mice pretreated with SRBC and CY. The subsequent injections of SRBC and Sal into tolerant mice did not increase the response under study. It is concluded that the effect observed is due to partial alteration of antigen-specific B cells of tolerant mice and this alteration cannot be explained by the lack of the regeneration of membrane immunoglobulins.     

Supressor activity of spleen cells during medically induced immunologic tolerance]

SRBC tolerance was induced in mice (CBA X C57BL/6) F1 by single intraperitoneal injection of 6 X 10(9) SRBC and of cyclophosphamide (100-200 mg/kg) in 44-46 hours. Spleen cells of tolerant mice obtained at various periods after the tolerance induction (in 12-26 days) failed to decrease their immune response to SRBC after administration to intact syngeneic recipients. Contrary to intact mice, tolerant animals were incapable of producing suppressor cells after a single SRBC immunization. Only when 3 additional injections of high SRBC doses (6 X 10(9)) were given to tolerant mice the spleen cells in them acquired the capacity to inhibit the immune response after administration to normal mice. It is supposed that the absence of suppressor cells in induction of the immunological tolerance by means of cyclophosphane was caused by the processes of clone elimination. Suppressor cells can originate in tolerant animals under the effect of intensive antigenic stimulation, this leading to enhancement of the tolerance state as a result of additional SRBC injections.     

Immune response and tolerance to S. typhi Vi-antigen in T-lymphocyte depleted mice]

Comparison of immune response to Vi-antigen in thymetomized letally irradiated and reconstituted with fetal liver cells mice and in control animals revealed no difference between the two groups. The absence of enchancement of antibody formation in T cell depleted mice favours thymic-independent regulation of immune response to optimal dose of Vi-antigen. The induction of cyclophosphamide tolerance to Vi-antigen did not depend on the presence of T cells: tolerogenic treatment was equally effective in T cell depleted mice and in control animals. Therefore cyclophosphamide tolerance was not due to the activation of T suppressors but to direct elimination of immunocompetent clones of B cells.     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.     

The immunological status of mice during the generation of cyclophosphamide-induced tolerance.

Tolerance to sheep erythrocytes (SRBC) has been induced by a combination of high doses of antigen and treatment with cyclophosphamide (CY). The influence of CY alone or in combination with SRBC has been investigated by using the treated animals as recipients for normal spleen cells. CY treatment appears to produce a mouse which is severely depleted of B cells. The injection of large doses of SRBC together with CY, in a schedule which induces tolerance, generates an environment which suppresses the production of antibody-forming cells by passively transferred normal spleen cells. However, transfer of cells from tolerant mice to irradiated mice failed to demonstrate the presence of suppressor cells.     

Role of T Cells in the Mitogen-Induced Proliferation and Polyclonal Antibody Response of Murine B Cells

The effect of thymus-derived lymphocytes (T cells) on the responsiveness of bone marrow-derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in a nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti-sheep erythrocyte (SRBC) IgM hemolysin plaque-forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti-SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC. Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti-SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti-SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.     

A reactivity to polyclonal stimulation of immunogenesis with salmozan after its repeated administration

The injection of 100 micrograms of salmozan (polysaccharide isolated from Salmonella typhi somatic O-antigen) or 50 micrograms of Escherichia coli lipopolysaccharide into mice induced a considerable increase in the number of antibody-forming cells in the spleen in response to the injection of sheep red blood cells (SRBC) 2-3 days later. This polyclonal effect was essentially weaker if the animals previously received 500 micrograms of salmozan (9-10 days prior to the injection of SRBC). The absence of reactivity was not linked with antibodies to salmozan or with some other serum factor. The lymphocytes of nonreactive mice proved to be capable of polyclonal response in the adoptive system, and at the same time the polyclonal response of intact lymphocytes to salmozan in the body of nonreactive irradiated mice was essentially weakened. The features making the above phenomenon similar to, as well as different from, the so-called "endotoxin tolerance" are analyzed.     

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

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

Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4+), T suppressor (Lyt-2+), and B cells

Continuous gastric intubation of mice with the T cell-dependent antigen sheep erythrocytes (SRBC) leads to a state of systemic unresponsiveness to parenteral SRBC challenge, a state termed oral tolerance. The systemic unresponsiveness of mice rendered orally tolerant to SRBC, however, is converted to humoral immune responsiveness by adoptive transfer of effector T contrasuppressor (Tcs) cells. In this study, the authors have isolated and characterized the Tcs cell subset, from the spleens of orally immunized mice, which abrogates oral tolerance. This Tcs cell is a novel cell type, which can be separated from functional T suppressor (Lyt-2+) and T helper (L3T4+) cells, and the effector Tcs cell exhibits a Lyt-1+, 2-, L3T4- phenotype. Furthermore, contrasuppression is not mediated by B cells, including those of the Lyt-1+ phenotype. Adoptive transfer of splenic Lyt-1+, 2-, L3T4- T cells from C3H/HeJ mice given oral SRBC for 21 to 28 days and splenic Lyt-1+, 2-, L3T4- T cells of C3H/HeN mice orally immunized for a shorter interval abrogated oral tolerance. Furthermore, separation of Lyt-1+ T cells into L3T4+ and L3T4- subsets by flow cytometry resulted in Lyt-1+, L3T4+ T cells with helper but not contrasuppressor function, whereas the Lyt-1+, L3T4- T cell fraction abrogated oral tolerance even though it was without helper activity. This Tcs cell subset was also effective when added to cultures of tolerized spleen cells derived from SRBC-fed mice. The effector Tcs cells are antigen-specific, because Tcs cells from SRBC-immunized mice reverse tolerance to SRBC but not to horse erythrocytes (HRBC), and Tcs cells from HRBC-immunized mice reverse tolerance to HRBC but not to SRBC. When splenic T3 (CD3)-positive T cells (Lyt-1+, 2-, and L3T4-) were separated into Vicia villosa-adherent and nonadherent subpopulations, active contrasuppression was associated with the T3-positive and Vicia villosa-adherent T cell fraction. Thus, a distinct Lyt-1+, 2-, L3T4- T cell subset that contains a T3-T cell receptor complex, which can regulate oral tolerance, is present in spleens of orally immunized mice.     

Immune responses of DBA/2 mice bearing melanoma tumors: Cell-mediated immune responses after challenge with vaccinia virus

Summary Cell-mediated immune responses in DBA/2 mice bearing melanoma tumors (TB-mice) were measured and compared to similar responses in mice without tumors (C-mice). Splenic lymphocytes from TB-mice had a reduced capacity to respond to both B and T-cell mitogens, but TB-mice responded to infection with vaccinia virus by developing a virus-specific cytotoxic T-cell response equal to that measured with splenic effectors prepared from virus-infected C-mice. NK-cell activity, as measured by the in vitro lysis of YAC-1 targets by splenic effectors, was significantly depressed in TB-mice but, after infection of the animals with vaccinia virus, was restored to levels equal to that measured with splenic effectors prepared from C-mice. Doses of vaccinia virus, strain WR which elicited vaccinia-virus-specific cytotoxic T cells or stimulated NK-cell activity, failed to elicit or stimulate cytotoxic effectors specific for S91-melanoma tumor cells.     

Suppressors of the graft vs graft reaction in tolerance to alloantigens

Immune response and suppressor cell activity of CBA (H-2k) mice made tolerant to allogeneic C57B1/6 (H-2b) heart graft were studied in graft-versus-graft reaction (GvGR). Intact CBA spleen cells inhibited response of (CBA X C57B1/6)F1 cells to antigenic stimulus (sheep red blood cells--SRBC), when injected together into lethally irradiated (CBA X C57B1/6)F1 mice. Spleen cells of tolerant mice were unable to decrease immune response of (CBA X C57B1/6F1 lymphocytes to SRBC and suppressed specifically the inhibition induced by intact CBA spleen cells. Spleen cells from tolerant mice were also capable of suppressing GvGR induced by CBA lymphocytes immune to C57B1/6 cells. Pretreatment of tolerant spleen cells with rabbit antithymocyte globulin and complement before adoptive transfer diminished markedly the suppression. The results obtained in the study suggest that suppression of transplantation immunity in this model is mostly due to T suppressor cells.     

The role of T-system immunity in reparatory regeneration of the bone tissue in animals

It has been demonstrated in experiments on mice (CBA X C57BL) X F1, thymectomized and irradiated by 800 R, with the haemopoietic system restored by bone marrow (B-mice) that in these animals, as compared with the controls, the changes in cellular immunity (inhibition of natural killer cells and stimulation of individual functions of the phagocytizing cells) are accompanied by considerable inhibition of osteogenesis. Compensatory regeneration of broken thigh-bone in B-mice is delayed by 5--10 days in various elements of the regenerated tissue in comparison with normal mice. Anatomical formation of the provisional callus in B-mice is not completed on day 21 and approaches a 14-day regenerate of the controls. The obtained results suggest the participation of T-system immunity in the reparatory regeneration of bone tissue.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Thymosin activation of T-killer precursors for hematopoietic stem cells]

Lymph node cells of normal CBA mice, syngeneic radiation chimerae CBA leads to CBA and B-mice after incubation with thymosin (fraction 5) were transplanted to sublethally irradiated recipients (CBA X C57BL) F1; the number of endogenic colonies in the recipient's spleen was then recorded. Thymosin was shown to increase the killer activity of the lymph node cells of normal mice CBA, syngeneic chimerae CBA leads to CBA, but not of B-mice. As suggested, TU-cells' subpopulation served as target cells for thymosin.     

Mechanisms of polyclonal tolerance induced by lipopolysaccharide and cyclophosphamide

The treatment of recipient mice with LPS from S. marcescens followed by the injection of CY 48 h later inhibited a subsequent antibody production against unrelated antigen (SRBC) and polyclonal mitogen (LPS from Br. abortus). Such a reactivity persisted for 2-3 weeks after treatment. It was shown that the number of Ig+ cells in the spleens of treated mice was decreased, while the population of spleen Thy-1.2+ cells remained unaltered. Cell-cooperative test revealed that the function of B cells, but not T cells, was inhibited by the treatment. There were no changes in DTH response to SRBC. Thus, a subsequent treatment of mice with LPS and CY led to B-cell deficiency. The nature of this phenomenon is presumably the same as the nature of CY-induced antigen-specific immunological tolerance.     

Xenogeneic antisera against T-lymphocyte receptors recognizing allogeneic transplantation antigens: Preparation and properties

Antireceptor sera ARS-1 (anti-CBA-anti-C57BL/6) and ARS-2 (anti-C57BL/6-anti-BALB/c) were prepared in a xenogeneic system by immunizing rabbits with alloimmune CBA anti-C57BL/6 and C57BL/6 anti-BALB/c lymphocytes and subsequent exhaustive absorption of rabbit immune sera with red blood cells, lymphocytes, liver cells, and serum from intact mice. In the cytotoxicity test, both ARS sera lysed only activated T lymphocytes of corresponding, but no other specificities, and did not affect intact lymphocytes. They did not inactivate in vitro the PFC-producing antibodies to SRBC or inhibit the immune response to SRBC and Viantigen of Salmonella typhi in adoptive transfer experiments. Pretreatment of intact CBA lymphocytes in vitro with ARS-1 plus complement suppressed their ability to induce lethal GvH disease in irradiated (CBA × C57BL/6)F₁, but not (CBA × BALB/c)F₁ recipients; similar pretreatment of C57BL/6 lymphocytes did not lower lethality among irradiated (CBA × C57BL/6)F₁ recipients. This method of preparing ARS may help to obtain highly specific antireceptor sera for regulation of the immune response to different antigens in outbred animals and in humans.     

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

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

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

Functional heterogeneity among the T-derived lymphocytes of the mouse. VII. Conversion of T1 cells to T2 cells by antigen.

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.