Allotype suppression of the responsiveness of adoptively transferred adult spleen cells induced by affinity prepared anti-allotype antibody.

In an adoptive transfer assay it has been shown that a form of allotype suppression can be induced in adult spleen cells. Use of SPF congenic mice, and either anti-allotype serum freed of IgM rheumatoid factor or affinity prepared antibody, has helped to eliminate the possibility of a role for non-specific factors in allotype suppression. Both memory-cell development and a conventional primary response are more susceptible to suppression than memory expression in a secondary response. Non-specific suppressive effects on isotypes other than that of the target allotype (CH) are present but are much smaller than for specific suppression.     

Allotype suppression in an adoptive transfer system in adult mice: the specificity and feedback effects of a monoclonal IgG3 anti-(Igh-1b) allotype antibody.

Using an adoptive transfer system in mice, an allotype-specific suppression has been induced by a monoclonal IgG3 anti-Igh-1b (Hyb 5.7) reagent. Suppression was specific for IgG2a (Igh-1b) and led to a compensatory increase of the Igh-1a response in irradiated mice reconstituted with allotype heterozygous (Igha/b) spleen cells. Suppression, which was not antigen-specific, lasted for at least 1 month after anti-allotype treatment.     

Adoptive transfer of immunity to Nocardia asteroides in nude mice.

Nude mice on a BALB/c background were adoptively transferred with unprimed spleen cells, Nocardia-primed spleen cells, or Nocardia-primed splenic T lymphocytes from syngeneic, heterozygous (nu/+) littermates. Two days later, these recipient mice and unmanipulated (control) nude mice were infected intravenously with a 50% lethal dose of Nocardia asteroides GUH-2 from an early stationary-phase culture. Antibody titers, spleen weights, percent mortality, and organ clearance of the microorganisms were measured at 3 h to 28 days after infection. Adoptively transferred nude mice had larger spleens and greater titers of anti-nocardial antibody 7 to 28 days after infection as compared with control nude mice. Adoptive transfer with either primed spleen cells or primed splenic T lymphocytes enhanced both the survival of recipient nude mice and their ability to eliminate N. asteroides from the liver and spleen. These data indicate that adoptive immunity to infection with N. asteroides can be transferred with either specifically primed spleen cells or splenic T lymphocytes. Thus, it appears that cell-mediated immunity and T lymphocytes are of uppermost importance in host resistance to nocardial infection.     

Antibody-mediated allotype suppression in adult mice: the role of antigen, effector isotype and regulatory T cells

It has been reported (Contemp. Top. Immunobiol. 1974. 3:41) that allotype-specific T suppressor cells can be induced after monoclonal anti-allotype treatment of neonatal (BALB/c X SJL)F1 (Igha/b) mice. Here we show that (BALB/c X CB20)F1 adult-derived spleen cells (SC) are, by contrast, potently suppressed by monoclonal allotype-specific reagents, (when transferred into irradiated BALB/c recipients) in the absence of primary T suppressor cell induction. Such suppression is only induced in activated B cells [exposed to lipopolysaccharide or sheep red blood cells (SRBC)], and is probably dependent on the isotype of the anti-allotype sera administered. For example, two independently produced IgG1 monoclonal reagents raised against the Igh-1b allotype were poorly suppressive or nonsuppressive, whereas an IgG3 and an IgG2a monoclonal antibody induced a 90% suppression of the target allotype in transferred adult SC. It was found that suppression was not due to a depletion of antigen-specific T cell help since: (a) the addition of SRBC-educated T cells did not break suppression and (b) suppressed SC were as good a source of T cell help as normal SC, in the response of virgin or memory B cell (Thy-1-depleted) responses to SRBC in vivo. Suppression was maintained in suppressed cells which had been rechallenged with SRBC after transfer into a second irradiated recipient, but was not induced in normal SC when these were admixed with an equal number from this suppressed SC population. These findings point to a possible mechanism for the regulation of B cell expression, through the formation of an antibody-Ig receptor complex at the surface of the B lymphocyte. After complexing the target cell is either deleted or inactivated. The response to SRBC was reduced or ablated for at least 70 days after treatment with a single dose of anti-allotype serum.     

The suppressive effect of carrier priming on the response to a hapten-carrier conjugate

Carrier-primed cells, having some of the properties of T lymphocytes, have been found to inhibit the primary anti-hapten response of spleen cells to challenge with a hapten carrier conjugate. The antibody response of CBA/Ig^b spleen cells in irradiated CBA (Ig^a) recipients was measured by means of a radioimmunoassay using ¹²⁵I-labeled anti-Ig^b antibody. It was found that the primary anti-2,4,6-trinitrophenyl(TNP) response to TNP-KLH (keyhole limpet hemocyanin) was suppressed in recipient mice primed with KLH as compared with similarly treated unprimed or bovine serum albumin-primed recipients. This suppression was transferred by injection of 10⁸ spleen cells from CBA mice primed with KLH seven days beforehand, but not by injection of serum from such mice. The suppressive effect was abolished by treating the carrier-primed spleen cells with anti-T cell sera and complement before transfer. Priming with KLH seven days beforehand had only a small suppressive effect on the response to an unrelated antigen, DNP-ovalbumin, but there was marked suppression if KLH was again administered with the unrelated antigen. It is considered that the suppressive effect is specific in induction, but nonspecific in expression and that it is a manifestation of a homeostatic mechanism limiting the extent of the immune response.     

Suppressor T cells in low zone tolerance. I. Mode of action of suppressor cells.

Low zone tolerance (LZT) to bacteriophage fd seems to be a type of tolerance which is primarily caused by suppressor T cells. The aim of this paper is to analyze their mode of action. For the induction of antigen-specific suppressor cells in hydrocortisone pretreated CBA mice, we use tolerogenic and immunogenic doses of antigen. Suppressor activity can be demonstrated upon transfer of spleen cells into normal syngeneic mice. After immunization these animals are unable to produce IgG antibody against phage fd, whereas the IgM response is not suppressed; The half-life of transferred suppressor cells in nonimmunized animals is 5--6 weeks. The target of suppression are unprimed T helper cells, whereas primed helper cells cannot be blocked. T helper cells become "resistant" to suppression 18--36 h after contact with antigen. Differentiation from unprimed B into B memory cells is unaffected, yet under suppression conditions persisting B memory cells are blocked in IgG production. The experimental data are incorporated into a model of LZT.     

Specific IgM Enhances and IgG Inhibits the Induction of Immunological Memory in Mice

The effect of priming mice with IgM anti-SRBC (sheep erythrocytes) together with SRBC or IgG anti-SRBC together with SRBC on the development and expression of memory cells was studied. Mice primed with specific IgM and SRBC showed a much more efficient secondary plaque-forming cell and serum antibody response after challenge with SRBC in an adoptive transfer system than did controls primed with SRBC only. The expression of this enhanced memory of IgM-primed spleen cells was counteracted by the high levels of internal IgG anti-SRBC (also the result of priming with IgM) when the mice, instead of being tested in adoptive transfers, were challenged directly. The antigen-specific feedback suppression of the primary antibody response by specific IgG antibodies was also seen to inhibit partially the development of memory cells. The suppressive effect on priming could be demonstrated both in adoptive transfer systems and after direct boost of the same mice that received the primary immunization. Both the IgM enhancement and the IgG suppression of memory cell development were antigen-specific, since no effect on the antibody response to a non-cross-reacting antigen, horse erythrocytes, was seen. The effect of these up- or down-regulations of immunological memory could be demonstrated after secondary injections as long as 90-280 days after priming.     

Hapten-specific tolerance in mice. II. Adoptive transfer studies and evidence for unresponsiveness in the B cells

The suppression fo the anti-NIP ((4-hydroxy-5-iodo-3-nitrophenyl)acetyl) plaque-forming cell (PFC) response elicited in mice by treatment with NIP-coated syngeneic erythrocytes could be transferred by spleen cells into irradiated recipients. This was evidenced by the lack of an indirect anti-NIP PFC response 7 days after cell transfer and challenge with NIP. FGG (fowl IgG). This state of specific unresponsiveness could be serially transferred by spleen cells into a second irradiated recipient. The hapten-specific suppression in the first recipient could be reversed by addition of normal spleen cells, but not by cortisone-resistant thymus cells. The lesion appears to be in the T cell-depleted population, since the suppression could be reversed by supplementing with T cell-depleted normal spleen cells. In vitro incubation of spleen cells from tolerant animals did not restore the capacity of these cells to produce an anti-NIP PFC response in irradiated recipients. In vitro incubation of normal spleen cells with sera from tolerant animals did not prevent these spleen cells from producing a normal anti-NIP PFC response in irradiated recipients. The adoptive secondary response response to NIP. FGG was inhibited by injection of NIP-coated syngeneic erythrocytes on the same day as the adoptive transfer of the NIP. FGG primed spleen cells and the challenging antigen. It would seem that NIP-coupled syngeneic erythrocytes, which are presumably poor stimulators of T cells, can suppress NIP-specific B cells, perhaps by gaining direct access to the surface of these cells.     

Allotype suppression in the chicken. IV. Deletion of B cells and lack of suppressor cells during chronic suppression

Embryonally induced allotype suppression in M-1,G-1 heterozygous chickens was stable for at least 18 months after hatching. Suppression was established rapidly since injection of antigen only 4 days after anti-IgM-1 antiserum failed to abrogate its effect. Injected chickens had undetectable serum levels (i.e. less than 40 micrograms/ml) of the suppressed IgM-1 and low (0.3-0.6 mg/ml) levels of the linked IgG-1 allotype. This correlated with a complete depletion of cells bearing the relevant IgM-1 allotypes and a compensatory increase in the alternative nonsuppressed IgM-1 allotype-bearing cells in the spleen, peripheral blood and bursa. Cell transfer studies suggested that suppression could not be attributed to allotype-specific suppressor cells.     

Permanent hapten-specific tolerance in B lymphocytes.

Tolerance to the hapten TNP was induced in mice congenic with CBA but bearing the Ig-b allotype (Ig-b mice). To induce a high degree of tolerance it was necessary to give five injections of TNP-sulphonate followed by an immunogenic challenge (alum precipitate of TNP-BSA with pertussis adjuvant). Lymph node or spleen cells from these mice were transferred, with or without an equal number of non-tolerant CBA spleen cells, to irradiated CBA recipients and these were challenged with a different TNP-protein conjugate. Anti-TNP antibody bearing the Ig-b allotype was then assayed separately from total anti-TNP, as a measure of the contributions made by tolerant and non-tolerant B-cell populations respectively. Tolerant lymph node cell did not depress the response of normal cells, nor did the normal cells 'break' the tolerance of the Ig-b population even when the latter had been treated with anti-T-cell serum and complement. No response was obtained from tolerant lymph node cells when the recipients were challenged at different time up to 12 weeks after transfer. By this time the control non-tolerant lymph node cells had also lost the capacity to respond. It is concluded that: (1) effectively permanent tolerance, which is not maintained by afferent mechanisms, can be induced in lymph node B cells; (2) B-cell tolerance can be greatly enhanced by immunogenic challenge; (3) spleen may contain a distinct population of B cells which is less susceptible to tolerance; and (4) the life-span of virgin lymph node B cells is probably less than 12 weeks.     

Cellular requirements for the in vitro induction of specific suppression of antibody responses by immune spleen cells

Feedback regulatory signals to an intermediary suppressor cell may provide the mechanism of the suppression of antibody responses which has been described in cocultures of primed (immune) and unprimed (normal) spleen cells. The active cell(s) in the unprimed spleen population is nonadherent to nylon wool, Sephadex G-10 and glass beads. Lymph node cells and cortisone-resistant thymocytes from normal animals act similarly to normal spleen cells in this coculture system. Spleen cells from homozygous nude mice, unlike their heterozygous thymus-bearing littermates, do not produce a high degree of suppression in coculture with immune spleen cells. These data strongly suggest that the normal cell which interacts with primed cells in the cocultures to produce suppression is of thymic origin. However, spleen cells from neonatally thymectomized mice are suppressive in the presence of spleen cells immune to sheep red blood cells. The unprimed cell(s) active in the suppression are sensitive (in the presence of complement) to antisera directed against the surface markers, Thy-1, Qa-1, Lyt-1, Lyt-2, and Ia. Most of the antiserum treatments abrogated the suppressive capacity of the normal spleen cells only partially. Only treatment with anti-Qa-1 and complement routinely eliminated the ability of these cells to suppress in the cocultures suggesting either low concentration of or inaccessible surface alloantigen on the active cells. Alternatively, more than one Qa-1-positive cell set from the unprimed population may be involved. It is postulated that there is at least one subset of Qa-1-positive T lymphocytes present in unprimed spleen and lymph node cell populations capable of participating in the suppression of specific secondary antibody responses when cocultured with spleen cells from specifically primed animals.     

In vitro studies of allotype suppression in mice

Long-term allotype suppression in (SJL x BALB/c)F₁ mice has been investigated in vitro, using a culture system which can be maintained over a period of at least 2 weeks. Spleen cells from (SJL x BALB/c)F₁ nonsuppressed mice, primed 2 to 5 months earlier, were cultured at a concentration of 3 x 10⁶ cells together with 10⁶ spleen or lymph node cells from suppressed or from nonsuppressed (control) mice and challenged in vitro with SRBC. Cultures were assayed on days 5, 8, 11, and 14 by the PFC assay, using specific anti-allotype sera to develop indirect plaques. Cells from suppressed mice were extremely efficient in preventing a “b” allotype response of the primed cells in vitro, even though the “a” allotype response in the same cultures was unaffected. A time lapse of approximately one week in culture was required before suppression was very obvious. The suppressive effect was abolished by treatment of suppressor cells with anti-θ serum. Evidence is presented that the suppressive effect is due to the production of a diffusible factor, rather than to a direct cell-cell interaction.     

Allotype suppression in the chicken. I. Generation of chronic suppression in heterozygous but not in homozygous chickens.

The injection of antibody directed against either the IgM-1 a or IgM-1 b allotype into heterozygous (M-l^a/M-l^b, G-l^g/G-lⁱ) B 14-line chickens produced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes in their serum, as quantitated by single radial immunodiffusion. Suppression of the IgM-1 and IgG-1 allotypes was associated with a compensatory increase in the alternative IgM-1 and IgG-1 serum allotype levels. This suppression was induced (a) by passive injection of anti-allotype antiserum into 13-day-old embryonal or neonatal recipients, or (b) by egg yolktransmitted antibody in chickens hatched from homozygous B 14 A (M-l^a, G-l^g) hens immunized against the IgM-l b of the homozygous B14C (M-l^b, G-lⁱ) rooster. Heterozygous chickens injected embryonally with anti-allotype antiserum were profoundly suppressed for at least 16 weeks after hatching, while neonatally injected chickens showed a gradual recovery of both IgM-1 and IgG-1 allotypes over the same period. Suppression of the IgM-1 b allotype could be induced in heterozygotes which had inherited the M-l^b allele either maternally or paternally. However, no suppression of either IgM-1 or IgG-1 levels could be detected in homozygous chickens injected with the relevant anti-allotype antiserum. Hence, allotype suppression only occurred in M-1 heterozygous chickens which had an alternative source of B cells available. The involvement of a B cell surveillance mechanism in allotype suppression is postulated and the possible role of suppressor cells is discussed.     

Functional differences between B cell-depletion techniques in removing memory B cells. Relevance to anti-erythrocyte autoantibody-specific suppressor cells

The efficiency of a number of B cell-depletion techniques was examined in a functional assay. CBA (Iga) mice were primed with rat erythrocytes and their spleen cells transferred, before and after B cell depletion, to mice of the allotype-congenic strain Igb. The recipients were challenged with rat erythrocytes and their anti-erythrocyte autoantibody response was measured together with the donor (Iga) and host (Igb) anti-rat erythrocyte antibody levels. Transferred unseparated cells suppressed erythrocyte autoantibodies and produced high levels of anti-rat erythrocyte antibodies. Transfer of panned, rosetted or nylon wool-passaged cells neither altered donor anti-rat erythrocyte antibody levels nor abrogated suppression. By contrast, passage of rat erythrocyte-primed B cells over Ig-anti-Ig-coated beads resulted in removal of donor rat-primed B cell activity and loss of suppressor cells. The B cell-depletion techniques were effective at removing B cells as judged by the reduced number of fluoresceinated anti-Ig-labeled cells among unbound cells although analysis on a fluorescein-activated cell sorter revealed that the most effective method was depletion on Ig-anti-Ig-coated beads. B cell depletion by panning removed the capacity of virgin but not primed cells to make adoptive antibody responses after transfer suggesting that the primed cells most efficient in adoptive transfer have a lower density of surface Ig than virgin cells. Treatment of donors with drugs which abrogated transferrable suppression of erythrocyte autoantibodies did not alter donor anti-rat erythrocyte antibody levels in recipients. It is considered that rat primed B cells are involved in the suppression of erythrocyte autoantibodies by acting as selective antigen-presenting cells for T suppressor inducer cells, rather than through an anti-rat erythrocyte antibody feedback mechanism.     

Allotype suppression in the chicken III. Analysis of the recovery from suppression by neonatally injected or maternal antibodies

Injection of M-1 (Cμ), G-1 (CμgM) heterozygous chickens on the day of hatch with anti-IgM-1 antiserum induced allotype suppression from which chickens recovered over a period of approximately 4 months. The suppression of the serum IgM-1 levels was matched by a decrease in the number of splenic and peripheral blood B cells bearing the relevant IgM-1 allotype, and a compensatory increase in the number of cells bearing the alternative nonsuppressed IgM-1 allotype. However, the proportion of IgM-1-bearing bursal cells was only marginally altered. The recovery from suppression was due to B cell recruitment and could be abrogated by bursectomy. Allotype suppression induced in ovo or maintained by repeated injections of anti-IgM-1 anti-serum resulted in chronic suppression and depletion of the relevant peripheral as well as bursal IgM-1-bearing cells. Antibody titers of the relevant allotype in partially suppressed chickens generally correlated with serum allotype levels without clonal restriction in antibody response of the suppressed allotype.     

Interactions between primed and unprimed cells in the regulation of in vitro antibody responses. I. Role of “plasma cells” as inducers of suppression

Specific immune suppression has been shown to be activated in culture by the interaction of primed and unprimed T cell subsets. The primed cell involved is found 8 days after immunization in spleen but not in lymph node or thymus cell populations. When the primed spleen cells were fractionated by nylon wool passage or anti-Thy-1 plus complement (C) treatment, prior to culture with unseparated unprimed cells, suppression was detectable only with primed B cells present in the co-cultures. Treatment of the primed spleen cells with anti-PC. 1 (an antiserum specific for plasma cells) plus C eliminated their ability to cooperate with either unseparated or T cell-enriched populations of unprimed cells in suppressing the antibody response of the co-cultures. These data are consistent with the hypothesis that antibody-secreting plasma cells activate suppressor T cell precursors in cell populations not previously exposed to antigen.     

Allotypic suppression of adult mouse spleen cells: cell differentiation, class restriction and allotypic exclusion

Allotypic suppression of adult mouse spleen cells has been investigated by treating the cells with anti-allotype serum directed against the Ig-1 specificities of the γG_2a immunoglobulin class. Suppression of precursor, memory and primed cells in the anti-sheep red blood cell response, assayed in terms of the number of plaque-forming cells producing γM, γG1 and γG_2b immunoglobulins has been investigated. The results are discussed in terms of the stage of differentiation of cells when class restriction and allotypic exclusion occurs. It is concluded that γM or γG commitment occurs at an early precursor stage, and that γG class and allotype restriction occurs sometime after contact of precursor cells with antigen.     

Rapid loss of feedback suppressor T-cell activity after priming in vivo.

T cells from antigen-primed mice have a diminished capacity to mediate feedback suppression when compared to T cells from unprimed mice. This was demonstrated using an in vitro model of B-cell induced feedback suppression in which spleen cells from mice primed with sheep erythrocytes (SRBC) activate feedback suppressor T-cell precursors to mediate suppression of the primed spleen cell response. The addition of splenic T cells from unprimed mice to cultures of spleen cells from SRBC-primed mice resulted in suppression of the secondary IgM and IgG anti-SRBC response. In contrast, no suppression was detected when T cells from mice primed with SRBC were added to the primed spleen cell cultures. The loss of suppression by T cells from primed mice was antigen-specific and was detectable by 24 hr after priming, coinciding with the appearance after priming of T-cell enhancing activity. The reduced suppressive activity could be due to changes in the active T-cell subset itself or to the appearance of cells or factors within the T-cell population that block or mask detection of feedback suppression. In either case, the present finding suggests that priming of a host not only activates feedback suppression induction mechanisms, but also rapidly affects the ability of the T-cell population to develop effective feedback suppression.     

The functions of immune T and B rosette-forming cells

From 7 to 35 days after CBA mice were primed with SRBC their spleens were removed and anti-SRBC rosettes were formed. The rosettes were purified from other spleen cells by velocity sedimentation at 4° and rosette-enriched, rosette-depleted and various control populations were injected into lethally irradiated CBA recipients. These were challenged with SRBC and their spleens analysed for direct (IgM) and enhanced (IgG) PFC 7 days later. Removal of RFC depleted the primed spleen cells of their capacity adoptively to transfer an immune response. This depletion was antigen-specific. Purified rosettes alone prepared 7–8 days after priming transferred significant (relative to controls) immune reactivity to the irradiated recipients. Both B and T RFC were present at this stage and the response was dependent upon cell collaboration between these two populations. Later in the primary response (35 days) purified rosettes transferred negligible immune reactivity. But these RFC (90 per cent B) collaborated with rosette-depleted cells to restore full reactivity. B memory lymphocytes (AFCP) form rosettes from 7 to 35 days after immunization but T memory cells only do so for a limited stage during the peak of the primary response. The majority of T memory cells probably never form rosettes in this system. It is suggested that most T RFC may be cells mediating delayed hypersensitivity or are `passive' rosettes.     

T cell-dependent suppression of antibody production. I. Characteristics of suppressor T cells following tolerance induction.

Specific immunological tolerance was induced in adult CBA mice by a single injection of deaggregated human IgG (dHGG). Spleen cells taken 7 to 42 days later, produced consistent suppression of a DNP-HGG collaborative antibody response on adoptive transfer into heavily irradiated recipients. Noncentrifuged F(ab')2 fragments of HGG were as effective as dHGG in the production of suppressor cells. Suppression was antigen-specific since HGG-tolerant cells failed to abrogate either a DNP-keyhole limpet hemocyanin collaborative response or antibody production to the noncross-reactive antigen, horse erythrocytes. Pretreatment of the tolerant cell population with anti-Thy-1 serum and complement reversed the suppressive effect. However, purified tolerant T cells obtained by passage through nylon wool or anti-Ig columns were less effective than the original spleen cells in mediating suppression. Analysis of the cell types appearing in the column effluents indicated that the reduction in suppressive activity is best explained by retention of T cells rather than macrophages. Different T cell populations, however, were retained on the two types of columns. In the case of anti-Ig columns, these consisted of Ly-2,3+, Ia+ effector cells, whereas nylon wool columns caused depletion of Ly-1,2,3+ cells which are known to act as amplifiers of suppression. Suppression could not be explained in terms of delay in differentiation of antibody-forming cell precursors since the effect persisted for up to 15 days after transfer of tolerant cells. The demonstration of a reduction in serum anti-DNP and anti-HGG antibodies excluded the possibility of antibody production in sites other than the spleen. A role for anti-carrier antibody-antigen complexes in mediating the effector phase of suppression was rendered unlikely by the finding that the suppressive effect of tolerant cells persisted in the absence of detectable anti-HGG antibody production. Effector T cells mediating suppression in this system were shown to bear the phenotype Ia+, Ly-2,3+ as judged by the effect of pretreatment with appropriate antisera and complement. They were spleen-seeking, but were not detected in the thymus or recirculating lymphocyte pool. Adult thymectomy failed to cause a significant reduction in suppressive activity by tolerant spleen cells indicating that at least a major component of the immediate precursors is not of recent thymic origin.