Induction of immune tolerance to thymus-dependent antigen (sheep erythrocytes) in the absence of T-cells]

The role of T cells in B cell tolerance induction to sheep red blood cells (SRBC) was studied in intact adult mice, in lethally irradiated mice injected with singeneic embryonic liver cells and thymocytes (TB-mice) and in animals functionally deprived of T cells--thymectomized, letally irradiated mice reconstituted with embryonic liver cells only (B-mice). Tolerance was obtained by treatment of mice with SRBC and cyclophosphamide (Cy). Cy-induced tolerance to SRBC was shown to be the result of the absence of specific T cells and partially of immunocompetent B cells. Suppression of immunoreactivity was observed not only in TB-mice but also in B-mice subjected to tolerogenic treatment. Splenocytes of tolerant TB-mice did not suppress the immune response of intact spleen cells to SRBC. The results obtained suggest the conclusion that B cells tolerance could be formed in absence of T cells.     

Effect of thymectomy on induction of immunologic tolerance in adult mice and on the recovery of initial immunoreactivity]

The tolerance to sheep red blood cells induced with cyclophosphamide became more profound and prolonged in mice thymectomized before or after the tolerance induction. The greatest immunocompetence depression was achieved when the operation was preformed 24 hours before the tolerogenic treatment. The results obtained confirmed the assumption that this form of tolerance was due to deficiency of a definite T-helpers clones.     

Mechanism of tolerance to VI-antigen of Salmonella typhi induced by cyclophosphane

High dose Vi-antigen treatment and injection of cyclophosphamide 46 to 48 hours later induced in mice a state of immunological unresponsiveness remaining stable in adoptive transfer. Only low amounts of the antigen were revealed in the blood and spleen of tolerant animals 2 to 3 weeks after the tolerogenic treatment. No T-suppressors were found in the spleen of tolerant mice--the cells of tolerant mice failed to suppress the immune response of normal lymphocytes when transferred together to the irradiated recipients, or to induce tolerance in normal mice. Normal spleen cells restored partially the immune responsiveness in tolerant animals. The results obtained suggest that cyclophosphamide tolerance was due to deletion or the long-term inactivation of the immunocompetent cells.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. I. Organ differences of tolerogen susceptibility and cellular sites responsible for the resistance.

Cellular sites of the tolerogen resistance in DDD mice against human IgG (HGG) were examined by reconstitution experiments in which cells of various lymphoid organs from tolerized mice were transferred into lethally irradiated syngeneic recipients with or without the supplement of an excess number of untreated T or B cells. It was shown that T cells but not B cells in the spleen and bone marrow-locating B cells were tolerogen resistant. Kinetic profiles of tolerance induction were compared among thymus, lymph node, and spleen T cells. Thymus cells fall into unresponsive state as early as 2 days after the tolerogen (tHGG) injection when only partial tolerance was observed in lymph node T cells. By 1 week of tolerogen treatment, the tolerant state was completed in both thymus cells and lymph node T cells, while spleen T cells showed marked resistance. Tolerance induced in thymus cells and spleen T cells was of relatively short duration and responsiveness was completely recovered by 5 weeks after the injection of tHGG. At this time lymph node T cells still showed hyporesponsiveness. The differences in tolerance inducibility were also shown among different lymphoid organs in tolerogen dose response. Lymph node T cells were very sensitive to tolerance induction, giving no response even by the injection of 0.01 mg of tHGG. Thymus cells were much less sensitive with the gradual loss of responsiveness by increasing the amount of tHGG. In contrast, spleen T cells showed gradual resistance with increasing amount of tHGG, indicating that some positive response was evoked in spleen T cells by a relatively high dose of tHGG. These results seem to suggest that the tolerogen resistance of spleen T cells may be due to their capability of showing positive response against the tolerogenic material. This was also suggested by the fact that the treatment with cyclophosphamide following the tolerogen injection diminished completely the responsiveness against the subsequent challenge immunization.     

IgM and IgG response to sheep red blood cells in mouse hepatitis virus-infected nude mice.

In nude mice which originally had no ability to respond to sheep red blood cells, an enhanced response to the same antigen with IgM-IgG switching was demonstrated during subacute infection with mouse hepatitis virus. IgM antibody-producing cells in the spleen were detected at days 2 to 6 after the antigen injection and IgG antibody-producing cells appeared at day 6 or later. The secondary IgG response, though not remarkable, was recognized after reinjection of the antigen 10 days after the first injection.     

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.     

Tolerance induction in mice by conjugates of monoclonal immunoglobulins and monomethoxypolyethylene glycol. Transfer of tolerance by T cells and by T cell extracts

The specific tolerance induced in mice by conjugates of human monoclonal IgG (HIgG) with monomethoxypolyethylene glycol (mPEG) was transferred to normal mice by spleen cells or a surface immunoglobulin negative (sIg-) Lyt-2+ subpopulation of these cells. Although transferable tolerance was demonstrable 6 to 14 days after treatment of the cell donors with tolerogen, the state of tolerance persisted in the treated mice for at least 43 days. Moreover, an extract prepared by freezing and thawing of the sIg- spleen cells obtained from mice 6 days after treatment with HIgG(mPEG)20 was capable of reducing (greater than 85%) the immune response of normal mice to heat aggregated HIgG. On the basis of these results, it is suggested that similar tolerogenic mPEG derivatives of xenogeneic monoclonal immunoglobulins (XIg) may prove to be useful therapeutic agents in man when administered before treatment with the unmodified XIg.     

The inhibitory effect of T-2 toxin on tolerance induction of delayed-type hypersensitivity in mice.

Mice injected intravenously with 1 X 10(9) sheep red blood cells (SRBC) showed no delayed-type hypersensitivity (DTH) response to SRBC and were unresponsive to DTH induction by sc injection of an optimal dose of SRBC. However, when treated with T-2 toxin, a mycotoxin, 2 days after the iv injection, mice became to show significant DTH response and to be responsive to the DTH induction by the sc injection. When the spleen cells of the mice receiving the iv injection were transferred to unsensitized syngeneic recipients, the DTH response of the recipients to SRBC was suppressed. However, the suppressor activity of the spleen cells was decreased by T-2 toxin treatment. By the iv injection, cell population of the spleen was increased and that of the thymus decreased. In contrast, by T-2 toxin treatment 2 days after the iv injection, cell population of the spleen was not increased and that of the thymus was markedly decreased. The ratio of theta-bearing cells was increased in the spleen by the iv injection. However, such increase was not observed after the T-2 toxin treatment. The ratio of Ig-bearing cells in the spleen was not changed by the iv injection and the T-2 toxin treatment after the iv injection. T-2 toxin seems to interfere with generation of suppressor cells for the DTH response.     

Deficiency in immunocompetence of mice cured from large MOPC-315 plasmacytomas by melphalan therapy

Summary Mice cured from large MOPC-315 tumors by a single dose of melphalan, 7.5 mg/kg, were examined for up to 60 days after the drug treatment (71 days after the tumor inoculation) for their ability to respond to mitogenic stimulation, specific and nonspecific antigenic stimulation and for their susceptibility to inoculation with an unrelated tumor, L10 lymphoma. The response of spleen cells from cured mice to mitogenic stimulation by phytohemagglutinin or concanavalin A was slightly depressed at an early stage after the drug treatment. The allogeneic response against C57BL spleen cells and the antibody response against sheep red blood cells (SRBC) of spleen cells from cured mice remained below normal levels during the whole observation period. The deficiency in response to antigenic stimulation was found to be due to impairment in T-cell function. Cured mice were also deficient in their response to SRBC immunization (antibody and delayed-type hypersensitivity responses) and were more susceptible to inoculation with an unrelated tumor, L10 lymphoma, than normal, noninoculated mice. On the other hand, spleen cells of cured mice developed a highly specific cytotoxic response against target MOPC-315 tumor cells and the cured mice were resistant to challenge with an otherwise highly tumorigenic dose of MOPC-315. Thus, cured mice remained deficient for a long period of time in their response to MOPC-315-unrelated antigens but, at the same time, they showed a potent specific antitumor immunity potential in vivo and in vitro.Presented in part at the Ninth European Immunology Meeting, September 14–17, 1988, Rome, ItalyThe contribution of S. Shoval is in partial fullfillment of a PhD Thesis     

Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis

APC exposed to TGFbeta2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8(+) T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8(+), but not CD4(+) T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.     

The effect of exogenous whole-body hyperthermia on humoral immunity]

We investigated the proliferative responses of spleen cells (SC) to polyclonal mitogens lipopolysaccharide (LPS) and pokeweed mitogen (PWM), immune responses to sheep red cells (SRC) in mice undergoing hyperthermia. There were increased proliferative responses of lymphocytes to PWM if we used mice having rectal temperature 42 degrees C. Thermal shock in mice was accompanied by suppression of immune response. If we used mice suffering from hyperthermia (43-44 degrees C) for 20 minutes; there were decreased proliferative responses of lymphocytes to PWM or LPS for 10-30 days. We observed low immune response to sheep red cells in mice for 5-20 days. The changes of immune response were not revealed on the 40th day after induction of hyperthermia in mice.     

Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells

Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment Jalpha18(-/-) mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jalpha18(-/-) mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When Jalpha18(-/-) mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, Jalpha18(-/-) recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4(+) iNKT cells. We conclude that CD4(+) iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

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

Mice subjected to tolerogenic treatment by sheep red blood cells (SRBC) and cyclophosphamide were immunized at various intervals (from 1 to 8 weeks after treatment) either by a single injection of 5 X 10(8) SRBC or by a double-injection of 1 X 10(6) SRBC. In control mice both immunization methods proved to be equally successful. In the experimental animals the immunological memory formation and/or its realization was destroyed to a greater extent and was restored more slowly than the capacity to the primary response.     

Induction of tolerance in athymic mice with an antigen which is highly immunogenic in euthymic mice.

Adult congenitally athymic (nu/nu) mice were found to be unable to respond to aggregated human γ-globulin (AHGG), the normally immunogenic form of HGG, unless first reconstituted with specific T cells. However, pretreatment of nude mice with AHGG prior to T-cell reconstitution resulted in the induction of unresponsiveness. This state of tolerance was specific since pretreated animals responded normally to the noncross-reacting antigens turkey γ-globulin or DNP-Ficoll. Transfer of spleen cells from nude mice pretreated with AHGG into normal littermates did not significantly affect a subsequent anti-HGG response of the recipients. Conversely, nude mice pretreated with AHGG and reconstituted with normal littermate spleen cells were hyporesponsive to challenge with AHGG. The results of these experiments are discussed in reference to various models for the induction of B-cell unresponsiveness.     

B Cell Tolerance

The mechanisms of B cell tolerance were studied in an attempt to learn whether B cells rendered tolerant are present in the immune system in a potentially responsive form. The author tested the in vitro anti-trinitrophenyl (TNP) antibody-forming cell (anti-TNP AFC) response to TNP-immunogens and polyclonal B cell activators (PBA) of spleen cells taken from mice injected with a tolerogen, TNP-carboxymethylcellulose (TNP-CMC). Spleen cells from mice injected 5 days previously with 10 μg of TNP-CMC did not respond to TNP-sheep red blood cells (TNP-SRBC), T-dependent (TD) antigen or TNP-Ficoll, T-independent (TI) antigen. However, the same spleen cells responded to PBA, lipopolysaccharide (LPS) of Salmonella enteritidis and purified protein derivative (PPD) of BCG. The results indicate that B cells specific for TNP are present in a potentially responsive form. Spleen cells from mice injected with 500 μg of TNP-CMC did not respond to either TNP-immunogens or PBA. The state of unresponsiveness to PBA lasted for 12 days after the tolerogen injection. Responsiveness to PBA reappeared within the short period of 2 days, whereas unresponsiveness to TNP-immunogens lasted much longer. Unresponsiveness to PBA was relieved considerably by treating tolerant spleen cells with the proteolytic enzyme trypsin before in vitro stimulation. These results indicate that B cells rendered refractory are present in the immune system in a potentially responsive form.     

Lipopolysaccharide-induced suppression of the primary immune response to a thymus-dependent antigen.

The immune response to a thymus-dependent antigen was depressed in vivo and in vitro in spleen cells from mice injected with LPS i.p. a few days before challenge with the antigen. Spleen cells from LPS-injected mice could, however, respond with increase DNA synthesis after activation with polyclonal B and T cell activators in vitro. The LPS-activated spleen cells could actively suppress normal cells in their response to the antigen sheep red blood cells. The suppressor cells contained in the LPS-activated spleens were most likely B lymphocytes, and the possible mechanism for their inhibitory function is discussed.     

Role of self carriers in the immune response and tolerance. III. B cell tolerance induced by hapten-modified-self involves both active T cell-mediated suppression and direct blockade.

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved in vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/+ littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cyclo) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclo-treated) donors.Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients.In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cyclo-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in vivo. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors.     

Functional activity of T and B lymphocytes of mice undergoing spontaneous loss of tolerance]

The functional activity of splenocytes and thymocytes of mice tolerant to sheep red blood cells was investigated one and four weeks after tolerance induction. The tolerance was achieved by cyclophosphamide. The immunocompetence of thymocytes was fully reversed in lfour week time. The functional activity of T and B lymphocytes of the spleen was also partially recovered four weeks after tolerance induction. Preliminary thymectomy weakened but did not prevent completely the immunocompetence of T cells of the spleen from being recovered. No Tsuppressants were found in the thymus or spleen of the tolerant animals.     

In vitro studies of the genetically determined unresponsiveness to thymus-independent antigens in CBA/N mice.

The X-chromosome-linked B lymphocyte defect of CBA/N mice has been studied in vitro by comparing the ability of (CBA/N X DBA/2)F1 (X-/X- X X+/Y) male (X-/Y) and female (X-/X+) spleen cells to respond to the thymus-independent antigen DNP (or TNP)-AECM-Ficoll. (CBA/N X DBA/2)F1 male spleen cells failed to generate significant in vitro anti-TNP antibody responses to DNP- or TNP-AECM-Ficoll, in contrast to spleen cells from F1 female (X-/X+) mice which responded normally to these T-independent antigens. Spleen cells from male F1 mice responded almost as well as F1 female cells to the thymus-dependent antigen, TNP-sheep red blood cells (TNP-SRBC) in vitro. Adding F1 male cells to F1 female cells failed to reduce the response of the latter to DNP-AECM-Ficoll, suggesting that the inability of F1 male cells to respond was not due to active suppression. The response of F1 male spleen cells to TNP-SRBC was not impaired by adding high concentrations of TNP-AECM-Ficoll indicating that the mechanism of unresponsiveness was not tolerance induction in all TNP-specific precursors. Lymphocytes from F1 male mice were capable of forming anti-TNP antibody after stimulation with lipopolysaccharide (LPS) in high concentrations; DNP-AECM-Ficoll had no effect on this polyclonal response. B lymphocytes from mice bearing only the X-chromosome of the CBA/N strain thus display a profound defect in B cell activation. This functional defect may represent either an inability of the defective B cells to be activated by thymus-independent antigens or the absence of a sub-class of B cells which respond to thymus-independent antigens.