Mechanism of neonatal idiotype suppression. II. Alterations in the T cell compartment suppress the maturation of B cell precursors

The cellular mechanism in neonatally suppressed BALB/c mice, which maintains the chronic suppressed state of the TEPC-15 idiotype in the antibody response to phosphorylcholine (PC), was investigated. Cells taken from these suppressed mice cannot transfer suppression to adult BALB/c or affect the in vitro response to PC of adult BALB/c spleen cells. However, spleen cells or T cells from neonatally suppressed mice given to neonatal animals induce chronic suppression of the TEPC-15 idiotype in the anti-PC response. Co-transfer of T cells from neonatally suppressed cells with normal T cells prevented the induction of suppression in neonates. Transfer of T cells from normal or keyhole limpet hemocyanin-primed BALB/c increased the expression of TEPC-15 idiotype in chronically suppressed mice, whereas T cells from neonatally suppressed were ineffective. These findings show that T cells in neonatally suppressed mice can affect the development of immature but not mature cells. The restoration of TEPC-15 expression in neonatally suppressed animals by normal T cells and the failure to induce suppression in neonates by co-transfers of T cells from normal and chronically suppressed mice demonstrate the profound role of an altered T cell compartment in sustaining chronic idiotype suppression.     

Immunoregulation by antigen/antibody complexes. I. Specific immunosuppression induced in vivo with immune complexes formed in antibody excess

Specific immune complexes, prepared at different ratios of antibody to antigen, were examined for their effects on the antibody response of BALB/c mice to the cell wall polysaccharide antigen (PnC) extracted from Streptococcus pneumonia R36a. Mice immunized with complexes formed in antigen excess developed a PnC-specific antibody response that was equivalent to that in mice injected with free antigen. On the other hand, mice injected with complexes formed in antibody excess developed very little PnC-specific antibody. Furthermore, administration of immune complexes (formed in antibody excess) resulted in suppression of the response to an immunogenic dose of PnC given concurrently or 1 day after injection of immune complexes but not when the antigen was given 1 day before injection of the immune complexes. Injections of free antibody (TEPC-15) also resulted in suppression of the response to antigenic challenge; however, suppression was greatest when the antibody was injected concurrently with the antigen, suggesting that the suppression was mediated through the formation of immune complexes in vivo. The suppression appears to be specific for the antigen (PnC), since in mice injected with TEPC-15/PnC complexes (formed in antibody excess) and challenged with PnC coupled to sheep RBC, only the response to PnC was suppressed.     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Idiotype shifts caused by neonatal tolerance to phosphorylcholine

The injection of as little as 0.5 microgram phosphorylcholine-(PC) conjugated mouse immunoglobulin into BALB/c neonates within 48 hr of birth results in complete unresponsiveness to PC for 3 to 4 wk. Thereafter, anti-PC responses can be detected in tolerized animals, but these responses differ significantly from those of normal BALB/c mice. First, the magnitude of responsiveness does not approach normal levels even 9 mo after birth. Second, although the initial responses as tolerance is broken can be T15+, idiotypic dominance is not established; instead, a heterogeneous T15- population eventually emerges, which includes clones with higher and with lower avidity than T15. Unirradiated unresponsive mice will help transplanted normal B cells to produce T15+ responses to thymus-dependent PC antigens. The responses of animals recovered from tolerance are stable upon adoptive transfer. We have, moreover, found no evidence of either loss of idiotype-specific T cell help or generation of suppression. Therefore, neonatal exposure to PC tolerogen can effect profound, permanent changes in the antigen-specific B cell compartment independent of any influence on conventional T cell regulatory mechanisms.     

Tolerance rendered by neonatal treatment with anti-idiotypic antibodies: induction and maintenance in athymic mice

Congenitally athymic (nude) mice of BALB/c background and their littermates were rendered unresponsive to the phosphorylcholine (PC) determinant by neonatal injection of anti-idiotypic antibodies. The kinetics of recovery from unresponsiveness were found to be similar for both groups when measured over a 20-week period. Spleen cells from suppressed, athymic mice did not respond to PC and were able to inhibit the response of normal cells to PC when tested in vitro. These results indicated that a population of specific suppressor cells, which may be responsible for induction and/or maintenance of unresponsiveness, can be generated in the absence of a thymic environment.     

Coexistence of antigen-specific and idiotype-specific suppressor T cells in mice injected neonatally with a mixture of antigen and anti-idiotype antibody

Specific tolerance to phosphorylcholine (PC) can be induced in BALB/c mice by neonatal injection with either pneumococcal C-polysaccharide (PnC) containing PC or anti-TEPC-15 idiotype (T15id) antibody which recognizes the predominant idiotype of anti-PC antibody of BALB/c mice. Suppressor T cells (Ts) induced after treatment with anti-T15id antibody react with the T15id and PnC-induced Ts cells appear to recognize PC. A brief incubation of anti-id-induced, T15id-specific Ts with PnC-induced, PC-reactive Ts resulted in complete cancellation of their suppressor functions. However, both types of Ts were present in mice neonatally injected with mixtures of PnC and anti-T15id antibody. Neutralization experiments using either PnC-induced or anti-id-induced suppressor T cells strongly suggest that only one of the Ts cell types is functionally dominant in those mice: most frequently, T15id-specific Ts cells. The suppressor function of the other population is detectable only when the predominant Ts cell population is removed by anti-id or monoclonal IgM anti-PC (SP45) plus complement. However, both suppressor activities are completely eliminated when one of the Ts populations is removed by adherence to either antigen or T15id. These results suggest that mice neonatally injected with a mixture of antigen and anti-id antibody possess both types of suppressor T cells, yet only one type is functionally dominant.     

Induction and mechanism of tolerance to bovine serum albumin in mice given total lymphoid irradiation (TLI).

BALB/c mice given total lymphoid irradiations (TLI) were injected i.p. with bovine serum albumin (BSA) in saline, and challenged with DNP-BSA in complete Freund's adjuvant 6 weeks later. The latter animals made no anti-DNP antibody response as measured by a modified Farr assay, but made a normal anti-DNP response after challenge with DNP-BGG in adjuvant. Normal mice or mice given whole body irradiation were not tolerized by the i.p. injection of BSA in saline. Spleen cells from unresponsive mice (TLI + BSA in saline) suppressed the adoptive secondary anti-DNP response of sublethally irradiated syngeneic hosts given BSA-primed T cells, DNP-BSA-primed B cells, and DNP-BSA in saline. The suppressor cells were antigen specific, and were inactivated by in vitro treatment with anti-Thy 1.2 antiserum and complement. The findings suggest that soluble antigens administered to mice after TLI evoke a state of tolerance that is maintained by antigen-specific suppressor T cells. A similar mechanism may be involved in the maintenance of tolerance to allografts. These findings may have important clinical implications for patients treated with TLI for lymphoid malignancies.     

Effect of passive administration of alloantiserum containing antibody against putative acceptor(s) for T cell-replacing factor (TRF) in the neonatal stage on development of B cell activity responsive to TRF

Previously, we showed that the antiserum raised in male (DBA/2Ha X BALB/c)F1(DCF1) mice (T cell-replacing factor [TRF]-low response animals) by immunizing them with activated B cells from BALB/c mice (TRF-high-responders) contained antibodies against putative TRF-acceptor site(s). We have now evaluated the hypothesis that neonatal treatment of mice with the above antiserum suppresses the development of B cells responsive to TRF. Male DCF1 mouse anti-BALB/c B-cell antiserum or normal DCF1 mouse serum as a control was injected into BALB/c mice within 24 hr after birth. In the antiserum-treated mice, no augmented primary immunoglobulin M (IgM) antibody responses to sheep red blood cells (SRBC) were observed under the conditions in which markedly augmented IgM anti-SRBC responses were induced in control BALB/c mice, suggesting that development of B cells reacting with male DCF1 mouse anti-BALB/c B-cell antiserum is suppressed by the neonatal treatment with the antiserum. Furthermore, the development of B cell activity responsible for helper factors derived from T cells, such as TRF, was markedly suppressed in the neonatally antiserum-treated mice, whereas activity of B cells capable of interacting directly with helper T cells through antigen-bridges was not significantly affected by the same treatment. Such suppression of the B cell activity could be induced only when the antiserum was administered within 48 hr after birth. Moreover, neonatal treatment of mice with the antiserum induced suppressed responsiveness of B cells to a T-independent type 2 antigen, TNP-Ficoll. Neither serum-borne suppressive serum components nor suppressor cells were detected by the system employed. These results support the hypothesis that TRF responsive B cells constitute a subpopulation distinct from the other B cells capable of cooperating with helper T cells via cognate interaction.     

Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

Modulation of the IgE immune response to BSA by fragments of the antigen. I. Suppression by free fragments and by fragments conjugated to homologous gamma-globulin

Nonimmunogenic peptic fragments of bovine serum albumin (BSA), Fraction Ia, suppressed immune response to BSA in mice. Splenic T lymphocytes from mice treated with these fragments suppressed the anti-DNP response in irradiated mice reconstituted with DNP-BSA-primed cells, indicating carrier-specific suppression. The conjugate of Fraction Ia with mouse γ-globulin (MGG) was found to be an effective suppressive substance but it did not induce suppressor T cells. B cells from mice given Ia-MGG were unresponsive to BSA when transferred to irradiated recipients along with either normal or BSA-primed T cells. Thus, unresponsiveness to BSA was mediated by either T or B lymphocytes, depending whether the inducing substance was a free fragment of the antigen or fragments conjugated to homologous γ-globulin.     

Specific suppression of the immune response by HGG tolerant spleen cells. I. Parameters affecting the level of suppression.

After adoptive transfer, the spleen cells from mice made tolerant to human gamma-globulin (HGG) specifically suppress the immune response of normal spleen cells. However, this suppressive activity in the spleen cells of tolerant mice is only present for a brief period after treatment with tolerogen. Spleen cells from animals injected 10 days earlier with tolerogen reduce the immune response of an equal number of normal spleen cells by 75%. Spleen cells from mice made tolerant 40 days previously are, however, no longer suppressive, even though they remain completely unresponsive. These data suggest that active suppression of antigen-reactive cells is not the mechanism responsible for maintaining tolerance to HGG, but rather is only transiently associated with the tolerant state. Further evidence in favor of the separation of the tolerant state from suppressive activity is that complete suppression of the normal spleen cell response requires either a high ratio of tolerant to immune competent cells or a delay in the antigenic challenge of the reconstituted recipients. By contrast, such manipulations are not required to demonstrate the complete unresponsiveness of the tolerant cells after adoptive transfer.     

T cell regulation of the thymus-independent antibody response to trinitrophenylated-Brucella abortus (TNP-BA)

We have previously observed a reduction of the T cell-dependent primary antibody response to dinitrophenylated keyhole limpet hemocyanin, and an enhancement of the T cell-independent response to trinitrophenylated Brucella abortus (TNP-BA) in BALB/c mice after treatment with total lymphoid irradiation (TLI). To elucidate the relative contribution of T and B cells to the enhanced T cell-independent antibody responses after TLI, a syngeneic primary adoptive transfer system was utilized whereby irradiated hosts were reconstituted with unfractionated spleen cells or a combination of purified T and B cells from TLI-treated and untreated control mice. Antibody responses of purified splenic B cells from TLI-treated BALB/c mice (TLI/B) to TNP-BA were enhanced 10-fold as compared with those of unfractionated (UF) spleen cells or B cells from normal (NL) BALB/c mice (NL/UF and NL/B, respectively). Splenic T cells from normal animals (NL/T) suppressed the anti-TNP-BA response of TLI/B by more than 100-fold. NL/T neither suppressed nor enhanced the response of NL/B. On the other hand, T cells from TLI-treated mice (TLI/T) enhanced by 100-fold the anti-TNP-BA response of NL/B, but neither suppressed nor enhanced the response of TLI/B. Thus, T cells can regulate the "T cell-independent" antibody response to TNP-BA. However, experimental manipulation of the T and B cell populations is needed to demonstrate the regulatory functions.     

Maturation of B cell subpopulations responding to phosphorylcholine

In this study, the order of appearance of B cell precursors responding to different phosphorylcholine (PC) antigens was investigated. Cells from neonatal BALB/c mice were transferred to male (CBA/N X BALB/c)F1 immunodeficient mice, and splenic fragment cultures were set up at different times after transfer. Because Xid F1 mice are unable to mount a primary anti-PC response, the time to prepare fragment cultures after cell transfer could be prolonged. This created an expanded maturation effect in which small differences in the response pattern of different precursor subsets are amplified. Splenic fragment cultures containing neonatal precursors were immunized with PG-TGG-HY (TD antigen), PnC (TI-2 antigen), or PC-TGG-LPS (TI-1 antigen), or with combinations of these antigens. The in vitro responses to these antigens were found to be additive, indicating the existence of different subpopulations. The detected precursors were also analyzed with respect to the T15 idiotype, which is the normally dominant idiotype of the response against PC in BALB/c mice. The analysis demonstrated a distinctly different maturation sequence of the three B cell subsets in which the order of appearance is: TI-1, TD, and TI-2 responding precursors. The T15 idiotype is expressed dominantly in all stages except the early stage of the TI-1 precursors.     

Carrier-induced tolerance to nucleic acid antigens.

BALB/c and SJL mice were treated with nucleosides-IgG1 as a tolerogen, before either primary or secondary immunization with nucleosides-keyhole limpet hemocyanin. Nucleoside-specific responses were measured serologically by a modified Farr assay, with either 14C-labeled denatured DNA or nucleosides-131-I-labeled BSA as test antigen. Specificity of the response was tested by hapten inhition experiments. Multiple doses of nucleosides-IgG1 tolerogen given before the primary or secondary immunization effectively suppressed the secondary and tertiary anti-nucleoside responses. The tolerogen did not suppress the response to an unrelated hapten-KLH conjugate. The IgG alone did not suppress the anti-nucleoside response of BALB/c mice to nucleosides-KLH. Single doses of tolerogen before the primary or secondary immunization were less effective. Residual antibody in partially suppressed BALB/c mice showed changes in specificity as compared to controls. Suppression of the secondary response of SJL mice was measured much more readily by binding of nucleosides-131-I-BSA than by binding of denatured DNA. This reflected an altered specificity of the residual antibody; in control animals, antibodies were directed against all four nucleosides, whereas the antibodies of partially suppressed animals were directed only against guanosine. Suppression of anti-nucleic acid antibody responses may have therapeutic application in the management of systemic lupus erythematosus.     

Mechanisms of tumor-induced immunosuppression: evidence for contact-dependent T cell suppression by monocytes.

BACKGROUND: The progressive growth of tumors in mice is accompanied by down-regulation of specific T cell responses. The factors involved in this suppression are not completely understood. Here, we have developed a model to examine the role of host immune effector cells in the inhibition of T cell function. In this model, progressive growth of a colon carcinoma line, CT26, is accompanied by loss of T cell response to alloantigens in both cytolytic and proliferation assays. MATERIALS AND METHODS: The CT26 tumor was inoculated into BALB/c syngeneic mice. Tumor growth, cytolytic T cell responses, lymphocyte proliferation, and flow cytometric analysis was performed in tumor-bearing animals 7 or 28 days after tumor inoculation. RESULTS: Spleen cells from tumor-bearing mice were found to suppress the proliferative response of spleen cells from normal mice to alloantigens. Examination of the spleen cell population by FACS analysis revealed an increase in the percentage of monocytes as defined by expression of CD11b, the Mac-1 antigen. Removal of the Mac-1-positive cells from the tumor-bearing hosts spleen relieved suppression of the tumor-bearing mouse spleen cell proliferative response to alloantigens, and addition of the Mac-1-positive enriched cells suppressed proliferation of normal T cells in response to alloantigens. Cell contact was required for this inhibition. CONCLUSIONS: Tumor induction of suppressive monocytes plays an important role in the general immunosuppression noted in animals bearing CT26 tumors. Identification of the mechanisms responsible for this effect and reversal of tumor-induced macrophage suppression may facilitate efforts to develop effective immunotherapy for malignancy.     

Ultraviolet irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway

Direct UV irradiation of dendritic cells and Langerhans cells reduces their Ag presenting ability. However, the effects of UV on CD11c(+) cells located distally to the point of irradiation are poorly understood. Three days after UV irradiation (8 kJ/m(2)) of BALB/c mice, bone marrow cells were isolated and cultured for 7 d with IL-4 and GM-CSF for the propagation of CD11c(+) cells. Bone marrow-derived CD11c(+) cells from UV-irradiated or nonirradiated mice were loaded with dinitrobenzene sulfonic acid and injected into the ear pinnas of naive BALB/c mice. After 7 d, the ears were painted with 2,4-dinitro-1-fluorobenzene and the ear swelling determined 24 h later. A reduced contact hypersensitivity response was found in mice injected with CD11c(+) cells from the UV-irradiated animals compared with those injected with cells from the nonirradiated animals. Further, a long-lasting suppression of the memory response to 2,4-dinitro-1-fluorobenzene was created. This suppressed response corresponded to increased IL-10 and PGE(2) secretion by freshly isolated bone marrow cells from UV-irradiated mice, and to increased myelopoiesis. The reduction in competence of bone marrow-derived CD11c(+) cells from UV-irradiated mice was not due to delayed maturation, as it was maintained upon LPS exposure prior to CD11c(+) cell purification. The UV-induced effect was reversed by the administration of indomethacin to mice prior to UV irradiation and could be reproduced by s.c. PGE(2). These results show that UV irradiation of mice can affect the function of bone marrow-derived CD11c(+) cells via a mechanism inhibitable by indomethacin; this pathway is likely to contribute to systemic UV-induced immunosuppression.     

Mechanisms of B cell tolerance. I. Tolerance to dextran B1355 induced with the oxidized dextran.

The bacterial dextran B1355, which is normally a potent thymus-independent immunogen, was made tolerogenic by oxidation. The injection of the oxidized dextran into BALB/c mice before, at the same time, or up to 4 days after the injection of the immunogenic form of the dextran resulted in a marked immunologically specific suppression of the number of anti-dextran antibody-forming cells found in the spleen. This suppression resulted from a direct inactivation of antibody-forming cell precursors rather than from either inhibition of antibody secretion or the exhaustive utilization of precursor B cells that have been observed in other tolerance systems. A substantial degree of tolerance was achieved after only a 1-hr in vivo exposure of the spleen cells to the tolerogen. At a dose of 1 mg of oxidized dextran per mouse, tolerance persised for at least 3 weeks. A complete recovery was apparent by 10 weeks. The stability of the tolerance was demonstrated by transferring tolerant spleen cells to irradiated recipients. The response in the recipient animals to an immunogenic dextran challenge remained suppressed. It appears that the tolerogenicity of the oxidized dextran is due to its ability to couple covalently with free amino groups in or near the receptor site of the cell membrane via the reactive dialdehyde groups of the dextran.     

The effect of Mycoplasma arthritidis superantigen on immunogenesis in transplacental infection

The immune responsiveness of the progeny of BALB/c mice, responsive to M. arthritidis superantigen (MAS), and C57BL/6 mice, nonresponsive to MAS, infected with M. arthritidis during the second half of pregnancy was studied. The investigation revealed that in responsive animals the proliferative response of spleen cells to MAS was suppressed with the level of response to concanavalin A remaining unchanged. The spleen cells of the test mice reacted to syngenic intact cells as to xenogenic ones and suppressed reaction to MAS and the production of interleukin 1 in the culture of spleen cells taken from the intact syngenic animals. The data obtained in this study suggest that after the infection of pregnant BALB/c mice with M. arthritidis immune tolerance to MAS developed in their progeny, which was accompanied by the induction of suppressor cells inhibiting the production of interleukin 1.     

From cell biology to immunology--a short trip

Immunologic memory and immunoglobulin allotype suppression are discussed as problems in Cell Biology. Memory, the ability of an animal after a first antigenic exposure to give a heightened and faster immune response upon a second exposure to the same antigen, is shown to be a property of bone marrow-derived cell lines. Expression of this memory depends on interaction with thymus-derived cells from either non-immunized or immunized mice. Chronic allotype suppression is described for the first time. It is initiated by in utero or neonatal exposure of (SJL × BALB/c)F₁ mice, allotype a/b, to anti b antibody. Suppression lasts for long periods and continues in irradiated recipients which have received lymphoid cells from suppressed donors. Selection against b allotype producing cell precursors can explain suppression.     

Differences in T15 expression of primary and secondary anti-PC responses with T cells from T15+ and T15- donors

The expression of T15 idiotype dominance with T cells from T15- mice was investigated. It was found that T15 dominance in the T-dependent response to phosphorylcholine (PC) could be generated by unprimed BALB/c B cells with T cells from T15+ and xid T15- mice. T15 dominance was also expressed when T15 neonatally suppressed BALB/c were used as T cell donors. With PC-primed B cells, however, T15 dominance was not established until 3 wk after adoptive co-transfer with T helper cells from xid NBF1 mice. To further study the different efficiencies of T cells for T15 dominance, limiting dilution analysis was performed. The data demonstrate that T15-specific T cell populations in BALB/c mice and NBF1 male mice differ in their precursor frequencies. In BALB/c mice, a frequent set of T15/M167-recognizing T helper cells is present; a corresponding frequent set of cells is absent in NBF1 males. This difference is likely to be one of the reasons why dominance is not immediately established after transfer of T cells from xid mice.