Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF). II. Monoclonal anti-cryptococcal TsF inhibits both phagocytosis by a subset of macrophages and transfer of contact sensitivity

Monoclonal anti-cryptococcal TsF (which inhibits phagocytosis by macrophages) and anti-picryl TsF use the same two circuits to block the transfer of contact sensitivity (CS). Both arm macrophages which then release a macrophage suppressor factor (MSF) when exposed to antigen. This MSF depresses the transfer of CS. The evidence suggests that a single molecular species of TsF (MW ca. 70 kDa), which bears an antigen-binding site and I-J determinant, is responsible for MSF production and inhibition of phagocytosis. Anti-cryptococcal TsF also arms the T acceptor cell which then releases nsTsF-1 after triggering with a specific antigen (SCPA). This nsTsF-1, which depresses the transfer of contact sensitivity, was authentic, as shown by its I-J positivity (in contrast to MSF) and its role in the production of nsTsF-2. As anti-picryl TsF also inhibits phagocytosis, it was concluded that anti-cryptococcal TsF, originally detected by the inhibition of phagocytosis, and anti-picryl TsF, originally detected by inhibition of CS, are functionally equivalent.     

Nonspecific inhibitor of DNA synthesis elaborated by T acceptor cells. I. Specific hapten- and I-J-driven liberation of an inhibitor of cell proliferation by Lyt-1-2+ cyclophosphamide-sensitive T acceptor cells armed with a product of Lyt-1+2+-specific suppressor cells

Lyt-1+2+ hapten-specific T suppressor cells (Ts) from mice injected and then painted with picryl or oxazolone derivatives produce hapten-specific T suppressor factors (TsF) in vitro. Stimulation by painting with contact sensitizer (which need not be specific) gives rise to Lyt-1-2+, I-J+, cyclophosphamide-sensitive T acceptor cells (Tacc). When the Tacc population is armed with TsF and then is exposed to specific antigen in the context of I-J-controlled determinants (antigen-presenting, haptenized spleen cells and Ts sharing the same I-J subregion), a nonspecific inhibitor of DNA synthesis (nsINH) appears in the supernatant. This inhibitor suppresses the primary DNA synthetic response to concanavalin A, lipopolysaccharide, and alloantigens in both syngeneic and allogeneic lymphocytes. The nsINH is only effective when added to lymphocyte cultures less than 8 hr after the stimulation with concanavalin A. The nsINH, however, affects neither primary nor secondary cytotoxicity in vitro. These data suggest the mouse immune system is capable of selective regulation of the response to specific antigen by the production of nonspecific soluble suppressor factor(s).     

T suppressor factor activity is due to two separate molecules. The Lyt-1(-)2+I-J+ cells of mice primed with antigen make an antigen binding molecule which is only active when complemented by cofactor made by Lyt-1+2(-)I-J+ cells

Mice primed with picrylsulfonic acid (PSA) and then painted on the skin with picryl chloride produce antigen-specific T suppressor factor (TsF). In contrast unpainted primed mice fail to produce active TsF. This is not due to the absence of the antigen binding part of TsF but to the absence of a cofactor. This cofactor is (a) antigen nonspecific and occurs in potassium chloride extract of normal spleen cells. It also occurs in the 24 hr supernatant of normal cells modified by haptenisation with picryl or the unrelated NP antigen (4-hydroxy-3-nitrophenylacetyl), and in preparations of conventional TsF (PSA/PCl) from painted PSA-primed mice; (b) bears I-J determinants; and (c) is produced by Lyt-1+2(-)I-J+ cells. The antigen binding molecule occurs alone in the supernatant of PSA-primed mice. It lacks I-J determinants and has a molecular weight around 35,000 and 75,000. It is produced by Lyt-1(-)2+I-J+ cells and is only active when complemented by cofactor. However, the complementation is genetically restricted and the restriction maps to the I-J subregion of the MHC.     

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF). I. Picryl and oxazolone-specific TsF, which inhibit transfer of contact sensitivity, also inhibit phagocytosis by a subset of macrophages.

Monoclonal and conventional cryptococcal-specific T suppressor factors (TsF) (also called TsFmp) depress phagocytosis by a subset of macrophages, while picryl- and oxazolone-specific TsF depress the passive transfer of contact sensitivity. This paper shows that these haptene-specific TsF also inhibit phagocytosis by a subset of macrophages and, using this assay, that the anti-haptene TsF resemble the anti-cryptococcal TsF in five respects: (i) the need for reexposure to specific antigen to trigger the release of TsF; (ii) genetic restriction in action; (iii) possession of an antigen-binding site; (iv) expression of I-J determinants; and (v) inactivation by reduction and alkylation. Purification of the anti-picryl TsF by sequential affinity chromatography indicates that the inhibition of phagocytosis is due to the TsF itself and not to a TsF-antigen complex. The TsF inhibits phagocytosis by a direct action as macrophages treated with TsF and exposed to antigen do not release a second factor which inhibits phagocytosis. These results and those of the accompanying paper indicate that the anti-cryptococcal and anti-haptene TsF are functionally equivalent, antigen-specific suppressor factors.     

Nonspecific T suppressor factor (nsTsF) cascade in contact sensitivity: nsTsF-1 causes an Ly-1+2- I-A+ immune T cell to produce a second, genetically restricted, nsTsF-2

We studied the mode of action of the nonspecific T suppressor factor (nsTsF-1) made in the picryl (TNP) system when T acceptor cells armed with antigen-specific TsF are triggered by antigen in the context of I-J. This suppressor factor does not inhibit the passive transfer of contact sensitivity directly, as shown by its failure to inhibit passive transfer by immune cells deprived of I-A+ cells. Its immediate target is an immune, antigen-specific, Ly-1+2-, I-A+ T cell. This cell, which may be regarded as a T suppressor effector cell (Ts-eff-2), produces nsTsF-2 when exposed sequentially to nsTsF-1 and antigen. This nsTsF subsequently inhibits the passive transfer of contact sensitivity. The action of nsTsF-2 is MHC genetically restricted. As the nsTsF-2 bears I-A determinant(s), this raises the possibility that it may act by combining with the recognition site for I-A on the T cell that mediates contact sensitivity.     

Requirements for suppressor T cell activation

Third-order (Ts3) suppressor cells are generated after conventional immunization. These cells, however, will not mediate suppressor cell function unless specifically triggered by an activating signal, termed TsF2. This report analyzes the mechanism of this TsF2-mediated triggering event. TsF2-mediated suppression is genetically restricted by genes in the I-J and Igh-V regions. The target of the I-J restrictions is a firmly adherent accessory cell, which appears to express I-J-related determinants. These accessory cells are sensitive to cyclophosphamide treatment and 500 R irradiation. In contrast, the target of the Igh-V restriction of TsF2 appears to be the Ts3 cell, which carries antigen-specific, idiotype-related receptors. The mechanism of suppressor cell activation appears to involve two stages. Presentation of I-J-restricted TsF2 by I-J-compatible presenting cells and a second step involving idiotype-anti-idiotype interactions between TsF2 and the Ts3 cell. I-J compatibility is not required with the accessory cell for Ts3 activation. Finally, we hypothesize that the anti-idiotypic determinants expressed on TsF2 can serve as an internal image of antigen, thereby permitting specific targeting of the factor.     

Murine interstitial nephritis. VII. Suppression of renal injury after treatment with soluble suppressor factor TsF1

We prepared soluble suppressor T cell factor (TsF1) from donor spleens harvested from mice primed with tubular antigen-derivatized lymphocytes to analyze both its functional interactions with a larger suppressor T cell network and its influence on the nephritogenic effector T cell response producing interstitial nephritis to a parenchymal antigen. Our findings indicate that TsF1 is antigen-specific, genetically restricted by I-J in its direct mediation of suppression, and capable of inhibiting the development of interstitial lesions. TsF1 also provides an inducing signal for the activation of effector Ts-2 suppressors following presentation by accessory cells. The induction of a Ts-2 effect, however, requires that the factor-presenting cell and the recipient of such cells share homology at I-J, and that the TsF1, the precursor Ts-2 cells, and the recipient of the Ts-2 effect share the same Igh-V allotype. Finally, the results of this current report clearly demonstrate a possible therapeutic role for soluble suppressor factors in the management of interstitial renal disease.     

A primary in vitro antibody assay for antigen-specific T-suppressor factor: cross-suppression of TNP-specific antibody responses by TMA-specific TsF1

We have previously shown that phenyltrimethylammonium (TMA)-specific, first-order suppressor T cells (Ts1) and soluble factors extracted from these cells (TsF1) can suppress delayed-type hypersensitivity (DTH) responses. The TsF1, as monitored in the DTH system, was characterized and found to be a single-chain, antigen-binding, I-J+, and Id+ molecule. To monitor TsF1 in an efficient manner, an in vitro antibody system was developed. The studies show that in vitro stimulation of naive A/J spleen cells with the thymic-independent antigen, Brucella abortus, to which TMA and trinitrophenol (TNP) or fluorescein (FL) are coupled (TMA-BA-TNP or TMA-BA-FL), induces significant numbers of anti-TNP or anti-FL plaque-forming cell (PFC) responses. The addition of TMA-specific TsF1 results in the cross-suppression of 30-50% of the total anti-TNP and FL PFC responses. This activity is antigen (TMA) dependent since suppression occurs only when the TMA ligand is present in the culture media. Analysis of the TNP-specific PFC responses in nonsuppressed cultures revealed that 20-35% of the PFC bear the cross-reactive idiotype(s) (CRI) normally associated with anti-TMA antibodies. In cultures containing TMA-TsF1, CRI+PFC are suppressed by 90-100% while the CRI-PFC are suppressed only by 10-30%. Our studies further show that an induction-phase, antigen-binding, CRI+, and I-J+ single-chain factor is responsible for the observed in vitro suppression. The possibility of utilizing this assay to monitor a variety of antigen-specific suppressor factors is discussed.     

Analysis of hapten-specific T suppressor factors: genetic restriction of TsF1 activity analyzed with synthetic hybrid suppressor molecules

We have analyzed the first-order suppressor factor secreted by an azobenzenearsonate (ABA)-specific T suppressor cell (Ts) hybridoma. Treatment of the factor with 5 mM dithiothreitol (DTT) yields two fragments with distinct phenotypes and functional capabilities. One fragment is bound by a monoclonal anti-I-J antibody, the other is not. Further, although neither molecular fragment by itself is sufficient to suppress an ABA response, a mixture of the two reconstitutes the suppressive activity. The I-J- portion of the first-order suppressor factor (TsF1) presumably guides the antigen specificity; activity of the ABA-specific Ts I-J- TsF1 factor can be reconstituted with an I-J+ subunit of a TsF molecule of either sheep red blood cell (SRBC) or ABA specificity. The genetic restriction for Igh-linked determinants of the ABA/SRBC hybrid TsF molecules is influenced by the I-J+ portion, regardless of the original antigen specificity of that molecule. The data support a two-subunit TsF model. Polyclonal ABA-specific TsF1 molecules appear to resemble the monoclonal factor in structure.     

A genetically restricted suppressor factor that requires interaction with two distinct targets

We have previously described a genetically restricted suppressor factor (TsF3) that suppresses the terminal phases of the contact sensitivity response. The activity of TsF3 is restricted by genes in the H-2 (I-J) and Igh complexes. This report analyzes the mechanisms responsible for these genetic restrictions. One cellular target of TsF3 is an I-J-bearing antigen-presenting cell population that is sensitive to low doses of cyclophosphamide. To elicit suppression I-J homology is required between this antigen-presenting cell population and the TsF3 donor. In contrast, the Igh-linked genetic restriction exists between TsF3 and an unprimed cell population present in the recipient. These findings suggest that under these experimental conditions TsF3 acts by bridging the APC with cells of the host. Finally, we demonstrated that nonspecific bystander or cognate suppression can be mediated by TsF3, provided specific antigen is present in the site of the ongoing T cell response.     

Mechanism of action of a T suppressor factor (TsF) in contact sensitivity: the T cell target for TsF activity in adoptive transfer of immunity is not effector cell

The passive transfer of contact sensitivity (CS) by immune cells into normal animals requires the interaction of two distinct Ly-1+ T cells, one which is Vicia villosa lectin (VV)-nonadherent, the other which adheres to VV. Functional deletion of either cell type abrogates the adoptive transfer of CS into normal animals, whereas VV-nonadherent cells alone can transfer CS into animals pretreated with cyclophosphamide (Cy). An antigen-specific T suppressor factor, designated TNP-TsF, inhibits the transfer of CS into normal adoptive recipients. TNP-TsF mediates its suppressive activity by inducing an I-J+ subfactor (designated I-J2) from the assay population by the interaction of PC1-F (a TNP-binding subfactor of TNP-TsF) with antigen-primed Ly-2+ T cells. This I-J+ subfactor then complements TNBS-F (an antigen-nonbinding subfactor of TNP-TsF) to form an antigen-nonspecific effector molecule which suppresses DTH responses in an antigen-nonspecific fashion. We report here that TNP-TsF suppresses the adoptive transfer of CS into normal animals but not into animals pretreated with Cy. TNBS-F + I-J2, the effector complex of TNP-TsF, also suppresses the transfer of CS into normal but not Cy-treated animals. When the Ly-1 immune cells were separated into VV-adherent and -nonadherent populations, the TNBS-F + I-J2 suppressor complex suppressed the functional activity of the VV-adherent cell population, but not the VV-nonadherent cells. This suppressive activity correlates with the need for VV-adherent cells in the transfer of CS into normal but not Cy-treated recipients. When an I-J+ molecule (I-J1) from an SRBC-specific TsF was used in place of I-J2 to form a suppressor complex with TNBS-F, this TNBS-F + I-J1 TsF suppressed the transfer of CS into both normal and Cy-treated recipients. This difference in functional suppressive activity correlated with a difference in target cell specificity: TNBS-F + I-J1 suppressed the VV-nonadherent TDTH cell, whereas TNBS-F + I-J2 suppressed the VV-adherent T cell of CS. Immune cells which are transferred under conditions which do not require the VV-adherent cell for transfer are not suppressed by TNBS-F + I-J2 or TNP-TsF, but are suppressed by the TNBS-F + I-J1.(ABSTRACT TRUNCATED AT 250 WORDS)     

TMA-specific first-order T-suppressor hybridoma. I. Characterization of the hybridoma-derived single-chain inducer suppressor factor, TsF1

Experiments described in this report will characterize a monoclonal phenyltrimethylammonium (TMA) specific, first-order T-suppressor factor (TsF1) produced by a T-cell hybridoma, 8A.3. The hybridoma expressed the Thy-1, Lyt-1, Lyt-2 antigens as well as cross-reactive idiotypic (CRI) determinants but did not express I-J encoded epitopes. It was also found to bear determinants recognized by a monoclonal antibody raised against single-chain GAT-specific TsF1. The hybridoma-derived factor was capable of suppressing primary in vitro trinitrophenol (TNP)-specific responses induced with the Brucella abortus antigen, conjugated with TMA and TNP haptens (TMA-BA-TNP). In addition, in vivo administration of 8A.3 culture supernatant resulted in the specific suppression of TMA-specific delayed-type hypersensitivity (DTH) responses. Analysis of this factor revealed it to be an induction-phase, antigen-binding, CRI+, and I-J+ single chain polypeptide. Our results represent only the second such described single chain, antigen binding, I-J+ suppressor factor derived from a monoclonal T-cell hybridoma.     

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.     

Information transfer between T cell subsets is directed by I-J+ antigen nonspecific molecules

T cell antigen-specific suppressor factors (TsF) consist of two distinct polypeptide chains: one that binds antigen (ABM) and one that bears I-J region markers (I-J+ chain). We studied the functional role of these two molecules in delivering the biologic message of suppression to its appropriate target cell. Two different biologically active TsF were used in these studies: TsiF, a T suppressor-inducer factor consisting of an ABM secreted by Ly-1 T cells (Ti-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Ji), which initiates the suppressor circuit by inducing an Ly-1,2 T cell; and TseF, a T suppressor-effector factor consisting of an ABM secreted by Ly-2 T cells (Te-ABM) and an I-J+ subfactor secreted by Ly-1 T cells (I-Je), which delivers the biologic message of suppression to the T helper (TH) cell. In both TsF, the ABM and I-J+ chain are noncovalently associated and can be easily separated. Both molecules must be present, however, for biologic activity of the TsF to be manifest. We studied the role of each chain in delivering these biologically active messages by constructing "hybrid" factors made from mixing the ABM from TsiF with I-J+ chains from either TsiF or TseF and determined which of these chains could reconstitute functional TsiF activity. Likewise, we mixed the AMB from TseF with I-J+ chains of TsiF or TseF to determine which I-J+ chain could reconstitute TseF activity. We found that I-J+ chain from TsiF (I-Ji) can reconstitute ABM from TsiF to form a functional TsiF capable of inducing suppression but cannot reconstitute ABM from TseF to form a functional TsiF capable of suppressing the activity of TH cells. Likewise, the addition of I-J+ chain from TseF to ABM from TseF can reconstitute its ability to suppress TH responses, but I-J+ chain from TsiF plus ABM from TseF has no effect on these TH cell responses. We did find, however, that this hybrid TsF composed of the ABM from TseF and the I-J+ chain from TsiF is capable of suppressing the Ly-1,2 Ttrans cell, the cell normally induced by the ABM + I-J+ suppressor inducer complex from T suppressor-inducer cells (TsiF).(ABSTRACT TRUNCATED AT 400 WORDS)     

I-J restriction of T cells that suppress in vivo antibody responses to Thy-1

The contact-sensitizing haptens dinitrophenyl (DNP) and oxazalone (Ox) act as helper determinants for antibody responses to Thy-1 when conjugated to donor thymus cells. The helper effect is transferrable from primed to naive mice with spleen cells, producing specific augmentation of in vivo PFC responses to Thy-1. The helper cells are hapten-specific and require associative recognition of hapten and Thy-1, excluding a role for nonspecific B cell activation. The phenotype of the helper cells is Thy-1+ and Lyt-1+2-. Antigen-specific suppression could be readily generated by using an inoculum of DNP-modified syngeneic RBC. T cells from these suppressed donors (Ts) were shown to abolish the helper effects of TH in adoptive transfer experiments in vivo. These Ts were characterized as Thy-1+ and Lyt-1-2+. A requirement for MHC compatibility at the I-J subregion was necessary between the Ts and the recipient to obtain a transfer of suppression.     

Dextran augments delayed-type hypersensitivity by interrupting one limb of the suppressor cascade

We have studied the immunomodulatory effect of dextran on the development of delayed-type contact hypersensitivity to a hapten in mice. Administration of an optimal dose of dextran 2 hours before applying picryl chloride to abdominal skin caused a twofold rise in the level of hapten-specific DTH. A study of the kinetics of development of DTH under the influence of dextran showed that comparable levels of response could be seen 2 days earlier in treated than in untreated mice, i.e., on the third day in contrast to the fifth day after sensitization. The peak of the responses, while greater in dextran-treated mice than in normal controls, remained the same at 5 days. Adoptive transfer studies revealed that comparable levels of DTH were conferred upon recipient mice by half the number of splenic cells from dextran-treated mice than that required from normal sensitized mice. Because several suppressor mechanisms are known to down-regulate DTH, we have studied dextran's effect on the neutralization of these systems as a possible explanation for its enhancing capabilities. Detailed examination was made of dextran's effect on the two suppressor T cells, Ts1 and Ts3, that act in tandem as well as its effect on the Ts1 and macrophage that work in combination. Both systems depress the efferent limb of DTH. We have found that dextran blocks the Ts1-macrophage pathway that controls DTH. Ts1 was found to arise normally in mice pretreated with dextran. Furthermore, Ts1 from dextran-treated mice produced TsF1 normally. However, we have found that dextran interferes with the production of macrophage suppressor factor (M phi-SF). Interference was partial when dextran was introduced during the interval in which macrophages were being armed with TsF1, and it was complete when dextran was put with pre-armed macrophages before they were triggered with antigen for production of M phi-SF. On the other hand, the Ts1-Ts3 limb of suppression remained unaffected by exposure to the immunomodulator. We found Ts3 arose normally in hapten-sensitized mice that had been pretreated with dextran. In addition, Ts3 became armed with TsF1 in vitro in the presence of dextran since the cells functioned properly to suppress mature DTH effector cells. Finally, TsF3 was able to act in vitro upon DTH effector cells despite the presence of dextran.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin-specific suppressor T cell factors

Murine antibody responses to heterologous insulins are controlled by MHC-linked immune response genes. Although nonresponder mice fail to make antibody when injected with nonimmunogenic variants of insulin, we have recently shown that nonimmunogenic variants stimulate radioresistant, Lyt- 1+2- helper T cells that support secondary antibody responses. However, the helper activity can not be detected unless dominant, radiosensitive Lyt-1-2+, I-J+ suppressor T cells are removed. In this paper we report that extracts of primed Lyt-2+ suppressor T cells contain insulin-specific suppressor factors (TsF) that are capable of replacing the activity of suppressor T cells in vitro. The activity of these factors is restricted by MHC-linked genes that map to the I-J region, and immunoadsorption studies indicated that they bind antigen and bear I-J-encoded determinants. Insulin-specific TsF consists of at least two chains, one-bearing I-J and the other the antigen-binding site. Furthermore, mixing of isolated chains from different strains of mice indicates that the antigenic specificity is determined by the antigen-binding chain and the MHC restriction by the H-2 haplotype of the source of the non-antigen-binding, I-J+ chain. Moreover, mixtures containing antigen-binding chain from allogeneic cell donors and I-J+ chain from responder cell donors have activity in cultures containing responder lymphocytes. This suggests that preferential activation of suppressor T cells, rather than differential sensitivity to suppression, results in the nonresponder phenotype to insulin.     

In vitro generation of suppressor T cells. Induction of CD3+, IgH-restricted suppressor cells

Previous studies of the immune response of C57BL/6 mice to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten determined that challenge with antigenic forms of hapten induces both immunity and suppression. The anti-NP plaque-forming cell response can be down regulated by an Ag-induced cascade consisting of three suppressor T cell subsets. These three populations, termed Ts1, Ts2, and Ts3 have been characterized to have inducer, transducer and effector functions, respectively. Although the functions of each of these subsets have been examined in vivo, the cellular requirements for in vitro Ts induction have only been investigated for the Ts3 population. The present study characterizes the cellular events that lead to the induction of the Ts2, suppressor transducer population. Culture of naive C57BL/6 spleen cells with Ts1-derived suppressor factor in the absence of exogenous Ag leads to the generation of Ts2 cells that mediate Ag-specific suppression of NP plaque-forming cell responses. Phenotypic analyses demonstrate that a CD3+, CD4-, CD5+, CD8+, and I-J+ precursor population is stimulated by TsF1 to become mature Ts2 cells that express CD3, CD8, and I-J but not CD5. Although previous studies have reported an essential role for B cells in the induction of other Ts populations, depletion of B cells from Ts2 induction cultures had no effect on Ts2 generation. Despite the absence of B cells in these cultures, the mature Ts2 cells were functionally IgH restricted. Studies with IgH congenic B.C-8 mice suggest that this restriction specificity was imposed by the idiotype-related determinants expressed on the TsF1, not the T cell genotype.     

Interaction of idiotype-specific T suppressor factor with the hapten-specific third-order T suppressor subset results in antigen-nonspecific suppression

The interaction between the third-order T suppressor (Ts3) cell and the idiotype (Id)-specific second-order Ts factor (TsF2) was studied in the phenyltrimethylamino (TMA) hapten system. The experimental system which we used allowed the independent analysis of induction and activation requirements of Ts3. The procedure consisted of inducing the Ts3 in vivo and activating the enriched T-cell populations containing Ts3 in vitro with TsF2. The suppressive potential was then tested in mice previously primed for delayed-type hypersensitivity responses which were also treated with cyclophosphamide to deplete Ts3 and other drug-sensitive Ts cell types. Using this experimental system, it was found that the Id-specific TsF2 was required for the in vitro activation of Ts3. Furthermore, the TsF2 activated only the homologous and not heterologous antigen-primed Ts3-containing T cells and moreover, the target of TsF2 was found to be the Ts cells bearing hapten-specific receptors. Once the TMA hapten-specific Ts3 was activated with TsF2, the ensuing suppression was antigen nonspecific. The data demonstrate that the Ts3 represents a final effector Ts cell type in the TMA system.     

T suppressor efferent circuit which affects contact sensitivity to picryl chloride: the late-acting, second nonspecific T suppressor factor bears I-A determinants which are responsible for the I-A genetic restriction in its interaction with its target cell

The T suppressor efferent circuit in the picryl (TNP) system, which inhibits the passive transfer of contact sensitivity, involves at least two antigen-nonspecific factors. The second nonspecific T suppressor factor (ns-2) bears I-A determinants of both the alpha and the beta chain as shown by affinity chromatography on immobilized anti-I-A monoclonal antibodies. Sequential absorption shows that the determinants of the alpha and beta chain occur on the same molecular complex. No absorption was obtained with anti-I-E antibody. There are two genetic restrictions associated with ns-2--the first is in its release from the second T suppressor efferent cell (on exposure to antigen) and the second is in its inhibitory interaction with its target cell. Both are MHC restricted and matching in I-A (but not I-E, or I-J) is sufficient. The question was asked whether the I-A of the ns-2 was directly responsible for the I-A genetic restriction in its action. F1 TsF was made in (H-2k X H-2b)F1 mice by injecting picrylated parental cells intravenously and triggering the release of ns-2 with the corresponding picrylated parental cells. Both I-Ak- and I-Ab-positive ns-2 were produced and were separated by affinity chromatography on immobilized anti-I-A monoclonal antibody. The I-A phenotype of these separated ns-2 of F1 origin determines the genetic restriction in their action; i.e., I-Ak+ ns-2 only inhibits passive transfer by H-2k cells and I-Ab+ ns-2 only acts on H-2b cells. In contrast, the I-A haplotype of the picrylated cell used to induce the Ts cell which makes ns-2 is unimportant. It was concluded that the I-A on the ns-2, and not a possible recognition site for I-A, serves as a restriction element. This finding suggests that ns-2 may act directly on the I-A-restricted T cell which mediates contact sensitivity.