Novel mechanisms of specific suppression of anti-hapten antibody response mediated by monoclonal anti-carrier antibody

The cellular mechanisms of the antibody-induced suppression of immune responses were analyzed in the keyhole limpet hemocyanin (KLH) system. Some of the monoclonal anti-KLH antibodies, like KLH-specific suppressor T cell factor (KLH-TsF), were demonstrated to suppress the anti-2,4-dinitrophenyl IgG but not IgM plaque-forming cell responses in a KLH-specific and H-2-restricted manner. The anti-KLH antibodies with suppressive activity reacted with, and in turn, stimulated the suppressor hybridoma (34S-281) with the anti-idiotypic receptor complementary to the idiotypic KLH-TsF of the inducer type. Moreover, because the suppressive activity of the anti-KLH antibody was completely abolished by the treatment of responding spleen cells with anti-Lyt-2 and complement, it was apparent that the suppressive antibody activated suppressor T cell pathways. The isotype or affinity of antibodies is not related to the suppressive activity, because suppressive and nonsuppressive antibodies possess a similar affinity belonging to the same Ig isotypes. It also has been demonstrated that the Fc portion is not the functional site, because the F(ab')2 fragment still has the activity. The antibody specificity is found to be important for determining whether the antibody is suppressive or not. In fact, anti-KLH 26, but not other antibodies without activity, recognizes the particular KLH epitope seen by KLH-TsF, and exclusively interacts with the anti-idiotypic suppressor T cells. Thus, the anti-idiotypic suppressor T cell receives signals both from the suppressive anti-KLH antibody and from KLH-TsF, and transmits the antibody-induced suppressor signals to the effector-suppressor pathway. The size of the repertoire of anti-idiotypic suppressor T cells involved in the suppression seems to be very limited, because only four out of 120 monoclonal anti-KLH antibodies were found to have suppressor activity. The possible mechanisms of the cell interaction mediated by the suppressive antibody are discussed.     

Antigen and receptor-driven regulatory mechanisms. VI. Demonstration of cross-reactive idiotypic determinants on azobenzenearsonate-specific antigen-binding suppressor T cells producing soluble suppressor factor(s)

The first detectable suppressor T cell (Ts) arising after i.v. administration of azobenzenearsonate- (ABA) conjugated syngeneic spleen cells to A/J mice has been studied for its receptor specificity and ability to produce soluble suppressor factor(s). This cell, termed Ts1, has a specific receptor for the eliciting antigen ABA, as demonstrated by selective binding to ABA protein- but not TNP protein-coated plastic dishes. The activity of ABA-Ts1 can be abrogated by treatment with anti-idiotypic antibodies made against anti-ABA antibodies of A/J mice (anti-CRI), indicating that these ABA-binding cells possess a surface receptor structure sharing idiotypic determinants with antibodies of the same specificity. Finally, soluble extracts from, antigen-adherent ABA-Ts1, but not nonadherent cells from the same spleen cell population, possess suppressive activity when assayed directly for afferent suppression or tested for their ability to trigger a second population of Ts (Ts2) in naive recipients. These findings demonstrate a close concordance between a T cell surface receptor, soluble T suppressor factors, and B cell derived antibody, all capable of direct recognition of the eliciting ABA antigen.     

Genetic and biological characterization of a T suppressor cell induced by anti-idiotypic antibody

The cellular and molecular characteristics of anti-idiotype-induced suppression have been investigated. We have shown that i.v. immunization of A/J or C.AL-20 mice with rabbit antibodies against the major cross-reactive idiotype on A/J anti-ABA antibodies induces splenic suppressor T cells (Ts) able to suppress T cell-mediated cytolytic and delayed-type hypersensitivity responses to ABA. In these studies, we compare the T suppressor activity manifested by anti-Id-induced suppressor cells with that described previously after conventional antigen priming. Results indicate that i.v. injection of anti-idiotypic antibodies primes for efferent level Ts; in contrast, i.v. administration of ABA-coupled cells induces afferent level suppressor cells. Soluble cell lysates, containing suppressor factor(s) derived from these anti-idiotype-induced Ts, can also mediate suppression of T cell immune responses in an efferent manner. Factor-mediated suppression is MHC-unrestricted and is also observed in mice pretreated with cyclophosphamide, suggesting that this activity is analogous to third-order suppression. Furthermore, this factor suppresses the T cell-mediated DTH and CTL responses in an antigen-nonspecific but Igh-restricted manner. These latter results suggest that the cellular elements conferring antigen specificity and Igh restriction are separate. The implications of these findings to the relationship between idiotypic elements, antigen-binding structures, and Igh restriction elements on immunoregulatory T cells are discussed.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Soluble factors in tolerance and contact sensitivity to 2,4-dinitro-fluorobenzene in mice. IX. A monoclonal T cell suppressor molecule is structurally and serologically related to the alpha/beta T cell receptor

Ts cells from mice tolerized with dinitrobenzene sulfonate produce a DNP-specific, MHC-restricted soluble suppressor factor (SSF) which regulates contact sensitivity to 2,4-dinitro-fluorobenzene. Previous studies have shown that the SSF-producing T cells and the soluble factor have the same hapten/MHC specificity suggesting that SSF may represent a secreted form of the Ts membrane receptor. The relationship between TCR proteins and SSF was investigated by examining the structural and serologic properties of a monoclonal DNP/H-2Kd-specific suppressor molecule produced by a Ts hybridoma. Reduction followed by alkylation abrogated the ability of the 3-10 molecule to inhibit transfer of contact sensitivity to 2,4-dinitro-fluorobenzene, indicating that intact disulfide bonds were a required structural property for suppression. Reduction of the 3-10 molecule followed by affinity chromatography on DNP-coupled Sepharose beads indicated that the 3-10 suppressor molecule is a dimer and that one of its chains binds to cell-free DNP. Serologic properties of the 3-10 molecule were examined by determining the ability of pan-reactive rabbit anti-TCR antibodies and anti-V beta 8 mAb KJ16.133 and F23.1 to adsorb suppressor activity from 3-10 culture supernatant and affinity purified 3-10 ascites material. All three reagents adsorbed the suppressor activity whereas control antibodies had no effect. When 3-10 material was passed through a F23.1-conjugated Sepharose affinity column, suppressor activity was recovered in the column eluate but not in the effluent fraction. When the 3-10 molecule was reduced and separated into its two chains (i.e., DNP-binding and non-DNP-binding chains), it was found that the anti-V beta 8 antibody F23.1-bound to the non-DNP-binding chain of the suppressor molecule. Collectively, these results indicate that the monoclonal 3-10 suppressor molecule is structurally similar to the alpha/beta TCR and suggest that the 3-10 molecule expresses a determinant encoded by the V beta 8 family of TCR genes. These results are consistent with our hypothesis that these suppressor molecules represent a secreted form of the TCR expressed on the surface of the DNP-specific Ts.     

Immunologic regulation of experimental cutaneous leishmaniasis. V. Characterization of effector and specific suppressor T cells

Resistant CBA mice infected with Leishmania tropica promastigotes develop concomitant and convalescent immunity against reinfection. This can be adoptively transferred by splenic and lymph node T cells with a threshold dosage of 1 to 2.5 x 10(7). The effector cells are of Thy-1+, Lyt-1+2- phenotype. The same immune cell population also adoptively transfers specific DTH to L. tropica, which is restricted by the major histocompatibility complex. On the other hand, highly susceptible BALB/c mice infected with L. tropica develop antigen-specific suppressor T (Ts) cells (previously shown to inhibit the induction and expression of DTH), which are capable, on transference, of reversing the healing of lesions induced by prior sublethal irradiation of BALB/c mice. As few as 10(6) of these T cells are effective in abrogating the potent prophylactic effect of 550 rad. The Ts cells are of Thy-1+, Lyt-1+2-, and I-J- phenotype. Sublethally irradiated and infected BALB/c mice produce antibody responses quantitatively and isotypically similar during the critical first 40 days after infection whether or not they are injected with 10(7) Ts cells (nonhealing vs healing). Thus, impairment of DTH and curative immune responses in BALB/c mice cannot be attributed to a helper function of these Thy-1+, Lyt-1+2- cells for the formation of suppressive antibody.     

Regulation of cell-mediated immunity in cryptococcosis. II. Characterization of first-order T suppressor cells (Ts1) and induction of second-order suppressor cells

Cryptococcosis patients frequently have high levels of cryptococcal antigen in their body fluids, and the levels of circulating antigen can generally be used to predict the patient's recovery, with high or rising antigen titers indicating a poor prognosis and low or decreasing levels a good prognosis. In a previous study, we reported on a murine model for studying the effects of cryptococcal antigen on host defense mechanisms. In that work, we demonstrated that an i.v. injection of cryptococcal antigen (CneF) into CBA/J mice, to simulate the antigenemia known to occur in human cryptococcosis, induced a population of T suppressor cells (Ts1) in the lymph nodes (LN). Upon adoptive transfer, the Ts1 cells specifically suppressed the afferent limb of the delayed-type hypersensitivity (DTH) response to cryptococcal antigen. In the present study, we show that the precursors of the Ts1 cells are sensitive to low-dose cyclophosphamide treatment and that the phenotype of the Ts1 cells is Lyt-1+, Ia+ (I-J+). LN cells from CneF-injected mice or a soluble factor derived therefrom can induce in the spleens of recipient mice a second-order suppressor cell population that suppresses the efferent limb of the DTH response. The cells that induce the second-order or efferent suppressor cells have the same phenotype as the cells that appear to suppress the afferent limb of the DTH response. The findings in this study indicate that a complex regulatory mechanism is responsible for the observed suppression of the DTH response in this infectious disease model. Furthermore, the suppressive circuit thus far defined for cryptococcal antigen is similar to the antigen-specific suppressor cell pathway outlined for certain chemically defined haptenic systems.     

Direct demonstration of specific suppressor T cells in the mice tolerant to type III pneumococcal polysaccharide: two-step requirement for development of detectable suppressor cells

Spleen cells from CAF1 mice made tolerant to type III pneumococcal polysaccharide (S3) with S3 coupled to syngeneic spleen cells (S3-SC) develop S3-specific suppressor T cells (Ts). These Ts could be demonstrated consistently only when spleen cells from tolerant mice were cultured in vitro with the specific antigen and the specific tolerogen. Spleen cells from normal mice cultured under the same conditions did not suppress the antibody response to S3. When different numbers of Ts were transferred to normal CAF1 mice, an unusual dose-effect pattern was observed. Maximal suppression of the S3 response occurred when relatively low numbers of Ts, 3 to 30 x 10(5) per recipient, were transferred, whereas larger numbers of cells, 150 x 10(5) per recipient, were not suppressive. These results indicate that a presumably T-independent antigen, S3, can activate antigen-specific Ts. These Ts exhibit unusual dose effects upon transfer and require both an in vivo induction period and in vitro activation for development of maximal activity. These latter observations suggest that S3 may activate a different population of T cells with suppressor function than do conventional T-dependent antigens. The loss of suppression observed when greater than optimal numbers of cells were transferred suggests that a second type of T cell, which has the ability to 'neutralize' the activity of S3-specific Ts, is also induced in the same spleen cell population.     

Hapten-specific responses to the phenyltrimethylamino hapten. IV. Occurrence of mechanistically distinct idiotypic suppressor T cells before the appearance of anti-idiotypic suppressor T cells induced by the monovalent antigen L-tyrosine-p-azophenyltrimethylammonium

We have previously shown that a single i.p. injection of the monovalent synthetic antigen, L-tyrosine-p-azophenyltrimethylammonium [tyr(TMA)] in complete Freund's adjuvant induces an anti-idiotypic T suppressor cell (Ts2) population that can be detected 6 wk later by its ability to shut down delayed-type hypersensitivity (DTH) specific for the TMA hapten. In this paper we present evidence that 2 wk after tyr(TMA) administration, a subset of Ts, termed Ts1, appears that is both functionally and phenotypically distinct from the late appearing Ts2 population. The early occurring Ts1 act only at the induction phase of the DTH response and can also suppress this response intrinsically. This latter point is in marked contrast to our previous observation that the tyr(TMA)-induced anti-idiotypic Ts2 fail to function intrinsically and can only be detected upon adoptive transfer into naive mice. Ts1 bear idiotypic receptors and are Ly-1+,2- in contrast to the anti-idiotypic Ly-1-,2+ Ts2 population. In addition, unlike the Ts2 population, Ts1 are comparatively nylon wool-adherent. Adsorption of Ts1 on either antigen- or idiotype-coated petri dishes indicate that the suppressor activity can be transferred only by antigen-binding cells. Cellfree factors prepared from spleens containing the Ts1 population can suppress DTH only if administered at the induction phase of the response, in contrast to the factors derived from the Ts2 population that act both at induction as well as effector phases, suggesting that Ts1 and Ts2 can function via soluble mediators. Finally, we show that when Ts1-bearing mice are primed and boosted for anti-TMA antibody formation, the resulting response was overall reduced with respect to the idiotype-positive and negative plaque-forming cells that differs from the Ts2-bearing hosts wherein the idiotypic component is preferentially suppressed. The appearance of Ts1 before the detection of Ts2 in the same experimental animals is discussed with reference to a normal physiologic sequence of events involved in suppressor pathways.     

Immunity to herpes simplex virus type 2. Suppression of virus-induced immune responses in ultraviolet B-irradiated mice

Ultraviolet B irradiation (280 to 320 nm) of mice at the site of intradermal infection with herpes simplex virus type 2 increased the severity of the herpes simplex virus type 2 disease and decreased delayed-type hypersensitivity (DTH) responses to viral antigen. Decrease in DTH resulted from the induction of suppressor T cells, as evidenced by the ability of spleen cells from UV-irradiated mice to inhibit DTH and proliferative responses after adoptive transfer. Lymph node cells from UV-irradiated animals did not transfer suppression. DTH was suppressed at the induction but not the expression phase. Suppressor T cells were Lyt-1+, L3T4+, and their activity was antigen-specific. However, after in vitro culture of spleen cells from UV-irradiated mice with herpes simplex virus type 2 antigen, suppressor activity was mediated by Lyt-2+ cells. Culture supernatants contained soluble nonantigen-specific suppressive factors.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XIII. Characterization of a third T cell population involved in suppression of in vitro PFC responses

The involvement of a third-order suppressor T cell population (Ts3) in the suppression of in vitro PFC responses was analyzed. It was shown that Ts2 effector-phase suppressor cells, induced by the i.v. injection of NP-coupled syngeneic spleen cells, require a third suppressor T cell population to effect NPb idiotype-specific suppression of an in vitro B cell response. This Ts3 population was shown to be present in NP-primed but not unprimed donors. The Ts3 population specifically binds NP and is Lyt-1-, Lyt-2+, I-J+ and bears NPb idiotypic determinants. The involvement of the Ts3 population in a suppressor pathway that requires recognition of idiotypic determinants is discussed.     

Functional analysis of cloned macrophage hybridomas. VI. Differential ability to induce immunity or suppression

We previously screened a series of macrophage hybridomas derived from fusion of P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells for their ability to induce I-J restricted Ts cell responses. One Ia+ macrophage clone (63) consistently induced Ag-specific, I-J-restricted Ts. To evaluate whether macrophage hybridoma 63 also induced delayed-type hypersensitivity (DTH) immunity, mice were immunized with hapten-coupled macrophage hybridoma cells. Hapten-coupled splenic adherent cells and control macrophage hybridomas induced significant primary DTH responses, whereas hapten-coupled macrophage 63 induced little or no immunity when injected into H-2 compatible hosts. However, macrophage hybridoma 63 specifically activated I-Ak, I-Ad, or I-Ed restricted T cell hybridomas/clones, in vitro in the presence of appropriate Ag. Three different strategies designed to eliminate suppressor cell activity were successfully used to demonstrate that hapten-coupled macrophage 63 could also induce in vivo immunity. First, after immunization with hapten-coupled macrophages, mice were treated with cyclophosphamide. Second, macrophage 63 was treated with anti-IJ idiotype antibody before 4-hydroxy-3-nitrophenyl acetyl hapten (NP) coupling. Finally, haptenated macrophages were injected into I-A compatible but I-J incompatible recipients. These protocols are known to inhibit the induction of Ts activity, thus these results indirectly suggest that there is stimultaneous generation of Ts activity in vivo. The latter hypothesis was tested in adoptive transfer experiments. Transfer of lymph node cells from NP-63 primed B10.BR (H-2k) mice induced immunity in naive 4R animals, whereas the same number of immune cells suppressed NP-induced DTH responses in 5R mice. The combined results indicate that a cloned macrophage line can activate both Th and Ts cells. Macrophages which induce Ts activity may be responsible for maintaining the balance of immunity vs suppression. The data support the hypothesis that IJ interacting molecules (IJ-IM) expressed on macrophages are critical for induction of suppressor cell activity.     

Prevention of granuloma development in the mouse by using T cell hybridoma products

The ability of an azobenzenearsonate (ABA)-specific suppressor T cell factor, a soluble extract from first order suppressor T cells (Ts1), and suppressor molecules produced by a long-term T cell hybridoma to regulate ABA-specific granuloma formation was studied. ABA-derivatized syngeneic spleen cells (ABA-SC) administered subcutaneously induced persistent delayed-type hypersensitivity (DTH) responses, detected by footpad swelling and hapten-specific granuloma formation by 72 and 96 hr after challenge with ABA-bovine serum albumin coupled to polyacrylamide beads (ABA-BSA-PAB). Soluble factors from ABA-specific Ts1 prevented DTH and granulomatous development after subcutaneous administration of ABA-SC. Moreover, the in vivo administration of a factor that is derived from a Ts1 functioning hybrid cell line induced a second set of suppressor cells (Ts2) that upon transfer to syngeneic ABA-primed mice were able to inhibit granuloma formation in the footpad, as well as in the gastrointestinal tract after challenge with ABA-BSA-PAB. These experiments demonstrate the dependence of the granulomatous reaction on T cell-mediated events, as well as the potential therapeutic efficacy of an antigen-specific suppressor T cell factor and a hybridoma T cell product in limiting antigen-specific granuloma formation in vivo.     

Suppressor T cell recognition of major and minor histocompatibility alloantigens: selected suppression of MHC-directed responses by minor alloantigen Ts

The induction of suppression by i.v. administered alloantigens in the murine host was analyzed as a model of the possible effects of blood transfusion on transplant survival. The results indicated that suppressor T cells (Ts) specific for minor histocompatibility alloantigens could be readily induced by the i.v. presentation of minor alloantigen-disparate spleen cells. In contrast, similar priming with cells differing solely at the H-2 major histocompatibility complex stimulated only positive T cell immunity, with no evidence of suppression. The induction of H-2 directed Ts activity could be accomplished only by i.v. priming with major plus minor incompatible donor cells, suggesting that suppressor cell recognition of minor alloantigens may have facilitated the generation of Ts against H-2-encoded major transplantation antigens. A role for minor histocompatibility antigens in the regulation of H-2-specific immunity at the effector level was also indicated. Ts induced by i.v. pretreatment with minor antigen-disparate donor cells not only suppressed the delayed-type hypersensitivity (DTH) response to the relevant minor alloantigens, but also inhibited DTH against unrelated H-2 alloantigens introduced during subsequent intradermal immunization. Suppression of H-2-directed T cell reactivity was specific in that the presence of the Ts-inducing minor alloantigens was also required and occurred only when the minor and unrelated major alloantigens were presented within the same inoculum, if not on the same cell surface. The capacity of Lyt-2+Ts or Ts-derived suppressive factors specific for one set of cell surface molecules to modulate responses to an unrelated group of surface antigens does not appear to represent a general phenomenon, because similar suppression of immunity to unrelated tumor-specific transplantation antigens by minor-specific Ts was not observed. These results are discussed with respect to the possible mechanism of H-2-directed suppression and the role of the I region in Ts recognition of antigen.     

Modulation of Schistosoma mansoni egg-induced granuloma formation. III. Evidence for an anti-idiotypic, I-J-positive, I-J-restricted, soluble T suppressor factor

Modulation of pathogenic egg-induced hepatic granuloma formation in chronically Schistosoma mansoni-infected mice is an immunoregulatory process. Adoptive transfer and in vitro studies have demonstrated that this suppression involves various T lymphocyte circuitries, and the participation of soluble suppressor factors has recently been noted in these systems. The present study has partially characterized a soluble suppressive activity extracted from the thymus glands of chronically infected mice (SmTsF) that modulates granuloma formation in acutely infected mice. The suppressive effect of SmTsF could be administered by multiple i.v. injections or by slow release from osmotic minipumps implanted i.p. Homologous and reciprocal transfers of SmTsF prepared from B10.A(3R) and B10.A(5R) donors indicated that SmTsF-induced suppression required homology between the donor and recipient at the I-J subregion of the major histocompatibility complex. Furthermore, the use of immunoabsorbents prepared with anti-I-Jk and anti-I-Jb sera demonstrated that CBA/J (H-2k) SmTsF was retained by, and could be recovered from, anti-I-Jk insoluble columns, but was unaffected by parallel treatment with anti-I-Jb sera. Subsequent immunoabsorbent studies showed that SmTsF did not bind to soluble egg antigenic (SEA) columns, and thus demonstrated a lack of idiotype, anti-antigen activity. However, columns prepared by using anti-SEA IgG from chronically infected syngeneic mice retained SmTsF suppressive activity, and it could be recovered by alkaline elution. These data are compatible with an interpretation that the suppressive activity expressed anti-idiotypic reactivity. Thus a thymus extract obtained from chronic, modulated, S. mansoni-infected mice can induce granuloma suppression in acutely infected mice. This activity is associated with an I-J determinant-bearing, possibly anti-idiotypic moiety or moieties. These observations further implicate some of the Ts cascades reported in other systems in the regulation of cell-mediated pathogenesis in chronic experimental schistosomiasis.     

Antibody inhibition of suppressor cell induction

The IJ genetic restrictions of suppressor T (Ts) cells are controlled by H-2-related determinants that are expressed on antigen-presenting cells. This has led to the hypothesis that Ts cells carry receptors for a self H-2-related ligand that is expressed on specialized antigen-presenting cells. We refer to this H-2-related ligand as the IJ interacting molecule. This report evaluates the ability of rabbit antibodies directed against idiotypes on monoclonal anti-IJ antibodies (the latter are presumably reactive with the Ts cell receptor) to bind IJ interacting molecule and to inhibit antigen presentation to Ts cells. Such anti-idiotypic reagents were prepared against T cell-reactive monoclonal anti-IJk and anti-IJd antibodies. The F(ab')2 fragments of these anti-idiotypic reagents blocked Ts cell induction. The inhibition was haplotype specific and mapped to the IJ region. The anti-idiotypic antibodies blocked the generation of Ts1, Ts2, and Ts3 cells. The cellular target of the blocking activity mediated by these anti-idiotypic antibodies is a macrophage. This was shown by using a cloned macrophage hybridoma line for both Ts induction and absorption of antibody activity. The combined data support the concept that macrophages express IJ interacting determinants that are responsible for Ts cell induction.     

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.     

Regulation of anti-hapten antibody response by chemically modified carrier antigen preferentially provoking delayed-type hypersensitivity (DTH). II. DTH-reactivity and carrier-specific suppression of anti-DNP antibody response induced by priming with dodecanoyl-BSA are mediated by functionally distinct T cell subpopulations.

Experiments were carried out to determine whether or not the cell populations involved in DTH and in the suppression of antibody response are identical. The effects of four treatments, i.e., adult thymectomy (ATx), X-irradiation, anti-mouse thymocyte serum (ATS) and hydrocortisone (HC) on the induction of DTH and on the carrier-specific suppression of antibody response were observed in mice immunized with chemically modified antigen, dodecanoyl-BSA (d-BSA), emulsified with complete Freund's adjuvant (CFA), with the following results: 1) DTH induced by immunization with D -BSA remained constant in adult thymectomized mice, whereas the suppression of antibody response was not inducible in these animals. 2) Injection of low doses of ATS caused the depression of DTH in mice primed with D -BSA, but did not affect the suppressive activities of their spleen cells. 3) Sublethal X-irradiation 1 week prior to D -BSA priming inhibited the generation of suppressor cells but did not affect the generation of cells mediating DTH. The suppressive effect was also abrogated by sublethal X-irradiation given 2 days after immunization with DNP-BSA (14 days after priming with D -BSA). 4) The treatment of animals with HC 2 days before the footpad challenge or immunization with DNP-BSA depressed the ability of animals to induce both DTH and the suppression of antibody response. However, the latter was more sensitive to HC than the former. In addition to these results, it was also found that D -BSA-primed spleen cells were capable of suppressing anti-DNP response, but not of inducing DTH-reactivity upon transfer to recipient mice. These results suggest that DTH-reactivity and the carrier-specific suppression of anti-hapten antibody response induced by injection of D -BSA are mediated by different cell populations.     

Suppression of antigraft immunity by preimmunization. III. Characterization of suppressor T cells involved in suppression of the efferent phase of DTH against alloantigens

Suppressor T (Ts) cells that can suppress delayed type hypersensitivity (DTH) against histocompatibility (H) antigens can be isolated from spleen and lymph nodes a few days after i.v. immunization of mice with irradiated allogeneic spleen cells. In this paper we investigated the suppression of the efferent phase of DTH to characterize the Ts cells involved, and to compare them with the afferent phase Ts cells that have been characterized in a previous paper of this series. The DTH against third party alloantigens that were not used for the i.v. suppressive immunization could be suppressed by presenting the third party alloantigens together with the original alloantigens in the challenge inoculum for eliciting the DTH reaction. Thus the ultimate suppressive effect by the Ts cells that are active during the efferent phase of DTH is nonspecific. This non-specific suppression of DTH to alloantigens has previously been found for the afferent phase Ts cells as well. For suppression of the efferent phase of DTH to alloantigens, a population of Lyt-1+2+ Ts cells appeared to be essential, just like in the suppression of the afferent phase of DTH to alloantigens. We did not find evidence for the involvement of cyclophosphamide-sensitive auxiliary Ts cells in suppression of the efferent phase of DTH. Also no evidence was found for H-2 or Igh-restricted activation and function of the Ts cells that were active during afferent and efferent phases of the DTH response to H antigens. In view of these similarities between afferent phase and efferent phase Ts cells we conclude that there are no arguments as yet to suppose that there is more than one type of T cells involved in the suppression of the afferent and efferent limb of DTH against H antigens.     

Antigen-specific T cell-mediated suppression. II. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine50 (GT)-specific T cell suppressor factor (GT-T8F) of suppressor T cells (T82) bearing distinct I-J determinants.

The induction of new suppressor T cells (Ts2) by suppressive extracts (TsF) from L-glutamic acid50L-tyrosine50 (GT) nonresponder mice was examined. Incubation of normal spleen cells with allogeneic GT-TsF for 2 days in vitro led to the generation of Ts2 cells able to suppress subsequent responses to the immunogen GT-methylated bovine serum albumin (GT-MBSA) in vivo. This induction occurred efficiently when TsF donor and target cells differed at all of H-2, including the I-J subregion. B10.BR (H-2k) GT-TsF, adsorbed on, then acid eluted from GT-Sepharose and anti-I-Jk [B10.A (3R) anti-B10.A (5R)]-Sepharose in a sequential fashion could induce BALB/c (H-2d) spleen cells to become Ts2 only if nanogram quantities of GT were added to the purified GT-TsF. This indicates a requirement for a molecule or molecular complex possessing both I-J determinants and antigen (GT)-binding specificity, together with GT itself, for Ts2 induction. The induced Ts2 are I-J+, since their function can be eliminated by treatment with anti-I-Jk plus C. These I-J determinants are coded for by the precursor of the Ts2 and do not represent passively adsorbed, I-J coded TsF, since anti-Ijk antiserum [(3R X DBA/2)F1 anti-5R] which cannot recognize the BALB/c (I-Jd) TsF used for induction still eliminates the activity of induced A/J (I-Jk) Ts2. These data provide further evidence for and information about the minimum of two T cells involved in antigen-specific suppressor T cell systems.