Ultraviolet-induced suppressor T cells and factor(s) in murine contact photosensitivity. I. Biological and immunochemical characterization of factor(s) extracted from suppressor T cells

Murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) is a highly specific, T-cell-mediated delayed-type hypersensitivity (DTH). Preexposure of the photosensitizing site to low doses of ultraviolet B(UVB) rendered mice unresponsive to challenge reaction. This unresponsiveness was associated with the generation of antigen-specific, afferent limb-acting, Lyt-1+2-,L3T4+ suppressor T cells (Ts-cps) in the spleen, thymus, and lymph node. Cell-free extract(s) obtained by freezing and thawing of these cells contained T-cell-suppressor factor (TsF) that inhibited the development of the induction phase of the CPS response to TCSA in vivo in an antigen-specific fashion. The treatments of TsF both with immunoadsorbent columns and with reduction and alkylation showed that the factor bore photoantigen-binding site(s), was reactive with monoclonal anti-I-Jd, anti-I-E alpha but not anti-I-Ad, and behaved as a single-chain factor containing both photoantigen binding and I-J molecules. By gel chromatography the majority of the suppressive activity was eluted in the fractions corresponding to molecular weights of 60-80 and 100-200 kDa. Our present study demonstrated clearly that UVB-induced unresponsiveness in the DTH reaction was mediated by a soluble suppressive factor derived from T cells.     

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.     

Anti-proliferative effect of IFN-gamma in immune regulation. II. IFN-gamma inhibits the proliferation of murine bone marrow cells stimulated with IL-3, IL-4, or granulocyte-macrophage colony-stimulating factor

A biphasic dose response curve was observed when the bone marrow-derived cell line FDCP1, used as an indicator line for IL-3 bioassays, was exposed to supernatants from some activated T cell clones but not others. The active component which inhibited proliferation at the higher supernatant concentrations appeared to be IFN-gamma, based on the following observations. 1) Only those culture supernatants which contained IFN-gamma gave a biphasic dose response curve; 2) with these supernatants, an anti-IFN-gamma mAb augmented the proliferation of FDCP1 cells at the higher supernatant concentrations; and 3) rIFN-gamma profoundly inhibited the proliferation of FDCP1 cells stimulated with rIL-3 or rIL-4. rTNF-alpha inhibited FDCP1 proliferation only to a modest extent, yet the combination of rTNF-alpha + rIFN-gamma provided greater inhibition than each agent alone. The proliferation of a second bone marrow-derived cell line, DA1, was not inhibited by rIFN-gamma or rIFN-gamma + rTNF-alpha when stimulated with rIL-3 or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Fresh bone marrow cells also showed a suboptimal proliferative response when stimulated with T cell supernatants containing IFN-gamma, and this response was augmented considerably upon the addition of anti-IFN-gamma mAb. Bone marrow cell proliferation was observed upon exposure to rIL-3, rIL-4, or rGM-CSF, and these responses were inhibited by rIFN-gamma; rTNF-alpha also produced a synergistic effect with these cells. Bone marrow cell colony formation stimulated by rIL-3 or rGM-CSF also was inhibited by rIFN-gamma. Colony formation in bone marrow cell cultures was not observed in response to rIL-4. Collectively, these results suggest that Th1 cells, which in addition to IL-3 and GM-CSF also produce IFN-gamma, may regulate hemopoietic cell proliferation and colony formation differently from the way Th2 cells do, which do not produce IFN-gamma.     

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.     

Cytokine regulation of bone marrow natural suppressor cell activity in the suppression of lymphocyte function.

Natural suppressor (NS) cells, which nonspecifically suppress immune responses, are present in the spleen following exposure to radiation, chronic graft-versus-host disease, or cancer and in normal bone marrow. A model system is described which allows the study of cytokines activating and inhibiting NS cells, cytokines mediating NS activity, and NS effects on cytokine synthesis. Recombinant interleukin-3 (rIL-3) and granulocyte-macrophage colony-stimulating factor (rGM-CSF) efficiently activated NS cells present in normal bone marrow and were effective at concentrations as low as 5 U/ml. At high concentrations, GM-CSF, but not IL-3, did not activate NS cells. Recombinant interferon-gamma (rIFN-gamma) blocked the activation of bone marrow NS cells by rIL-3, but did not down-regulate NS cells once activated. The NS cells secreted one or more soluble suppressor factors, which blocked IL-2 synthesis and also inhibited IL-2-dependent T cell proliferation in the presence of excess IL-2.     

Downregulation of helper T cells by an antigen-specific monoclonal Ts factor.

The findings of previous studies in this laboratory demonstrating that conjugates of human monoclonal (myeloma) IgG (HIgG) and monomethoxypolyethylene glycol (mPEG) were able to induce in mice antigen-specific tolerance and CD8+ suppressor T (Ts) cells were confirmed in the present study. An extract (TsF) of a nonhybridized clone of Ts cells (viz., clone 23.32), which had been derived from spleen cells of mice tolerized with HIgG(mPEG)26, was shown to possess antigen-specific suppressive activity. This monoclonal TsF was able to specifically suppress in vitro antibody formation only if it was present from the beginning of the culture. From the results of the cellular dissection of the system used it was concluded that (i) the TsF had no effect on fully differentiated primed B cells or plasma cells, and (ii) the TsF inactivated carrier-primed Th cells when the culture contained concomitantly naive CD8+ T cells, accessory cells, and antigen. These data support the view that the monoclonal TsF exerted its downregulating effect on Th cells only if it could first interact with a CD8+ T cell, in the presence of accessory cells and antigen.     

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.     

The suppressor T cell induced by syngeneic splenic cell antigen down-regulates hapten-specific cytotoxic T cells by elaborating a factor inhibitory for IL2-dependent cell replication

An in vitro study has been made of the mechanism by which a suppressor T cell, that is induced in lymph nodes by a syngeneic splenic cell antigen, prevents generation of cytotoxic T cells specific for hapten-altered self antigens. When popliteal lymph node cells exposed in vivo to syngeneic splenic cells were immunized in vitro with heat-treated syngeneic TNP-coupled thymocytes and excess helper factors, the Ts remained inactive. In this condition the exposed popliteal lymph node cells routinely demonstrated approximately twice the CTL response developed by lymph node cells from normal mice. Nevertheless, when triggered in vitro by splenic antigen on either X-irradiated B or T cells, the exposed but not the normal lymph node cells exhibited reduced hapten-altered self-specific CTL responses. Furthermore, T cells within spleen cell-exposed popliteal lymph node cell populations when reexposed to splenic T cells made a factor that was found to be suppressive of CTL generation by normal lymph node cells in vitro. The nondialyzable T-cell suppressor factor (TsF) did not appear to act on lymph node precursor CTLs, nor on helper T cells but instead acted at the level of utilization of helper factors in the development of CTLs. In an examination of the effect of TsF on cellular replication, TsF was found to be nontoxic for CTLL-20, an IL-2-dependent T cell, and it did not hinder the uptake of IL-2 by receptor blockade of this cell. Nevertheless, the replication of CTLL-20 that is IL-2 driven was diminished in the presence of TsF. Similarly, TsF was found to be inhibitory for T-cell proliferation stimulated by mitogen but had no effect on a B myeloma cell proliferative response. Thus, TsF appears to act as an inhibitor of a T cell's capability to replicate despite the presence of the stimulus for replication, namely, IL-2.     

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.     

Relationships between antigen-specific helper and inducer suppressor T cell hybridomas

Ts1, or inducer suppressor T cells, share many phenotypic and functional characteristics with helper/inducer subset of T cells. In order to evaluate the relationship between these cell types, we made a series of new Ts1 hybridomas by the fusion of Ts1 cells with the functionally TCR alpha/beta-negative BW thymoma (BW 1100). Three Ts1 hybridomas (CKB-Ts1-38, CKB-Ts1-53, and CKB-Ts1-81) were established that express TCR and produce Ag-specific suppressor factors constitutively, thus making it possible to study the nature and specificity of Ag receptors, MHC restriction, and lymphokine production by the Ts1 hybridomas. Results presented in this report demonstrate that all the Ts1 hybridomas described here express CD3-associated TCR-alpha beta. These three Ts1 hybridomas recognize Ag (NP-KLH) specifically in a growth inhibition assay and this recognition is restricted by IE molecules. Two of the hybridomas also produce IL-2 or IL-2 and IL-4 upon Ag-specific activation. Thus, by these three criteria the Ts1 hybridomas appear indistinguishable from Th cells. These three Ts1 hybridomas, however, release suppressor factors (TsF1) in the supernatant that suppress both in vivo DTH and in vitro PFC responses in an Ag-specific manner. Like the TsF1 factors characterized previously, the suppression mediated by these factors are Igh restricted and lack H-2 restriction. These factors mediate suppression when given in the induction phase but not during the effector phase of the immune response. The TsF1 factors are absorbed by Ag (NP-BSA), and anti-TCR affinity columns and the suppressor activity can be recovered by elution. The data are consistent with the interpretation that Ts1 inducer-suppressor T cells are related to Th cells; the feature that distinguishes these cells is the ability to produce Ag-binding factors that specifically suppress immune responses.     

Mechanisms of contact photosensitivity in mice. IV. Antigen-specific suppressor T cells induced by preirradiation of photosensitizing site to UVB

Mice were successfully contact photosensitized with 3,3',4',5-tetrachlorosalicylanilide (TCSA) plus black light irradiation. Pre-exposure of the photosensitizing site (ca 5 cm2) to UVB (280 to 320 nm; 400 mJ/cm2) rendered mice unresponsive to a challenge reaction. Cell transfer experiments demonstrated that the spleens from the nonreactive mice contained suppressor T cells (Ts) that were antigen-specific and that blocked the afferent limb of contact photosensitivity to TCSA. To exert suppressive functions, Ts required another population of cyclophosphamide-sensitive T cells that resided in the spleens of nonsensitized mice. The results provide evidence that UVB-induced aberrant homeostasis of the skin caused a marked suppression of immune system that is associated with the generation of Ts.     

Reconstitution of an inactive antigen-specific T cell suppressor factor by incubation of the factor with prostaglandins

Experiments were performed to test the hypothesis that prostaglandins are crucial to the ability of an antigen-specific T cell suppressor factor to deliver a suppressive signal. In the system employed, T suppressor cells release an antigen-specific factor (TsF) that suppresses the ability of effector cells to transfer contact sensitivity (CS) skin swelling responsiveness to adoptive recipients. Culture of TsF-producing cells in the presence of indomethacin caused production of an inactive TsF that could be reconstituted by incubation of this inactive factor with low concentrations of certain prostaglandins such as PGE2 or PGE1. Subsequently, nearly all the prostaglandins were removed by dialysis, and the reconstituted TsF then acted as an antigen-specific suppressor of CS effector cells. Neither the inactive factor nor prostaglandins were suppressive alone. Furthermore, the prostaglandins are crucial to the constitution of TNBSA-F, the non-antigen-binding subunit of the TsF that probably delivers the ultimate suppressive signal. These results provide a new type of antigen-specific role for prostaglandins in immunoregulation and indicate that simple, local, hormonal molecules in physiologic concentrations can have a crucial and long-lasting role in constituting the suppressive activity of antigen-specific regulatory macromolecules released by suppressor T cells.     

Functional analysis of antigen-nonspecific T-cell suppression. I. Effect of mitogen-induced T suppressor cells on helper-T-cell clones

The effect of mitogen-induced nonspecific suppressor T cells (Ts)2 on T-helper-cell activity was investigated using isolated clones of murine T-helper cells as targets. TNP-self-reactive Thy1+, Ly1+ T-cell clones were isolated after continuous culture of T cells derived from picryl chloride-sensitized mice and were characterized by their ability to proliferate in an antigen-specific and MHC-restricted manner. In addition, selected T-cell clones were found to produce both interleukin-2 (Il-2) and T-cell replacing factor (TRF), lymphokines characteristic of helper T cells. Concanavalin A (Con A)-induced Ts cells inhibited the antigen-specific proliferation of these helper-T cell clones in a noncytotoxic manner even in the presence of exogenous Il-2. This implied that failure to proliferate was not merely due to an inability of these clones to produce Il-2. The kinetics of suppression also suggested that early T-cell activation signals were not affected. Furthermore, coculture experiments indicated that while proliferation could be severely inhibited, the actual secretion of lymphokines such as Il-2 and TRF by the T-helper clones was not. Our data suggest that nonspecific Ts modulation of proliferation versus helper factor production are under separate control in cloned T-cell populations, with lymphokine secretion remaining intact in the presence of Con A-induced Ts cells.     

Induction of T cell responses in nonresponder mice by abolishing suppression with monoclonal antibodies recognizing A region-controlled, T cell-specific determinants

Mouse strains carrying the kappa allele at loci A beta, A alpha, E beta, and E alpha are nonresponders to lactate dehydrogenase B (LDHB) and to allotypic determinants of IgG2a myeloma proteins (for example, UPC10 used in this study). The nonresponsiveness to these antigens is caused by T suppressor (Ts) cells that prevent antigen-primed T helper (Th) cells from proliferating. We demonstrate here that monoclonal antibodies specific for an A region-controlled molecule selectively expressed on T cells (A-T) are capable of inducing anti-LDHB and anti-UPC10 responses of primed T cells from nonresponder strains. A monoclonal anti-J antibody that cross-reacts with the A-T molecule also induces responsiveness, whereas another J-specific antibody that lacks this cross-reactivity fails to do so. The mechanism of response induction is blocking of the interaction between the Ts cell or its factor (TsF) and the target of suppression, the antigen-specific Lyt-1+2- (Th) cell. The blocking occurs at the level of the Ts cell and the TsF. The data indicate that Ts cells and TsF carry a unique, A region-controlled molecule that is not only functionally analogous but also serologically similar to the J molecule.     

The immune response and the eye. I. The effects of monoclonal antibodies to T suppressor factors in anterior chamber-associated immune deviation (ACAID)

We report the effects of two monoclonal antibodies (mab) specific for murine T suppressor (Ts) factors (TsF) in anterior chamber (AC)-associated immune deviation (ACAID), as induced by AC inoculation of TNP-coupled syngeneic spleen cells (TNP-Spl). One mab (14-12) is specific for Ts effector factor and can block the induction of Ts cells in ACAID if given before or after AC injection of TNP-Spl. The other mab (14-30) is specific for Ts inducer factors and blocks suppression only after given after TNP-Spl. We also studied the surface phenotype of the Ts cells induced by AC injection of TNP-Spl. We show that at least two cells are required for the adoptive transfer of suppression in TNP-ACAID. One is Lyt-2+ and 14-12+, the other is I-J+. These Ts cells have the surface phenotype of Ts effector cells as seen in other systems. These results indicate that mab which bind TsF in other systems affect Ts cells in TNP-ACAID, and that the Ts cells induced in TNP-ACAID are only of the Ts effector type.     

Hapten-specific responses to the phenyltrimethylamino hapten. V. A single chain antigen-binding I-J+ first-order T suppressor factor requires antigen to induce anti-idiotypic second-order suppressor T cells

We have previously shown that a single i.p. injection of the monovalent antigen, L-tyrosine-p-azophenyltrimethylammonium in complete Freund's adjuvant induces a Ly-1+2-, idiotype-bearing, and antigen-binding first-order T suppressor (Ts1) population. We showed that soluble factors extracted from these cells could suppress delayed-type hypersensitivity responses if administered at the induction phase of the response. In this paper we additionally characterize the suppressor factor, TsF1, with respect to its biologic, serologic, and chemical properties. The studies show that the TsF1 is neither allotype nor H-2 restricted and can induce anti-idiotypic T suppressor cells (Ts2), but it requires the presence of antigen to do so. The factor binds antigen, bears I-J encoded determinants, is resistant to reduction and alkylation, and elutes as a single chain factor after adsorption onto monoclonal anti-I-J antibody-coupled Sepharose beads in the presence of dithiothreitol (DTT). This is in marked contrast to TsF2 (derived from Id-specific Ts2-containing spleen cells), which lost its suppressive activity after reduction and alkylation, and behaves as a two chain factor after adsorption and elution from anti-I-J-coupled beads in the presence of DTT. The TsF1 is discussed with respect to the properties of it and those of TsF1 from other similar idiotype-dominated antigen systems.     

Suppressor T cell growth and differentiation: evidence for induced receptors on suppressor T cells that bind a suppressor T cell differentiation factor

T suppressor cell differentiation factor (TsDF) induces the differentiation of alloantigen-primed suppressor T cells (MLR-Ts) to expression of their effector function, i.e., to active TsF production. The initial activation stimulus to Ts is provided by alloantigen binding; after this binding, Ts are functionally responsive only for a period of hours to the additional stimulus provided by TsDF. The present studies addressed the possibility that MLR-Ts responsiveness to TsDF reflects the induced and transient display of TsDF-binding receptors. TsDF receptor expression was investigated by determining the capacity of TsDF-responsive MLR-Ts to adsorb TsDF activity and to respond to that TsDF pulse by TsF production. Primed Ts populations that were alloantigen restimulated for 8 hr adsorbed TsDF in a cell dose-dependent fashion and produced TsF in response to that adsorption, whereas alloantigen-stimulated naive cells or primed but nonrestimulated cells neither responded to nor bound TsDF. Primed and restimulated L3T4-Ly-2+ but not L3T4+-Ly-2--enriched T cells bound TsDF. TsDF adsorption was saturable and time and temperature dependent. Glutaraldehyde fixation did not prevent TsDF adsorption by restimulated MLR-Ts, whereas pronase treatment abolished their TsDF-binding capacity. Kinetic analyses demonstrated that the capacity to bind TsDF developed rapidly after alloantigen reexposure, with maximal binding within 8 hr, followed by rapid decay with loss of TsDF binding by 36 hr. The kinetics of TsDF-induced TsF production correlated precisely with those of TsDF binding. These observations provide strong evidence that TsDF affects primed alloantigen-reactive Ts by interaction with antigen-induced and transiently expressed cell surface receptors. TsDF-receptor binding is then the stimulus for expression of Ts effector function.     

Ligand-receptor relationships in immune regulation

The relationship between ligand, idiotype-bearing ligand-binding T suppressor cells (Ts), and antiidiotypic Ts is discussed. The suppressor pathway involves the activation by ligand of first-order idiotypic Ts (Ts1) which elaborate idiotype-bearing T suppressor factors (TsF). TsF readily induced second-order antiidiotypic TS2 cells. The genetic restrictions imposed on the immune system once perturbed by antigen are evaluated.     

Purification and analysis of an antigen-specific suppressor factor from a T cell hybridoma specific for phenyltrimethylamino hapten

The synthetic monovalent antigen L-tyrosine-p-azophenyltri-methylammonium (tyr (TMA)) induces in A/J mice, a cascade of regulatory T cells in the absence of any detectable effector function (e.g., CTL, delayed-type hypersensitivity, etc.). An important component of the activated T cells is a first order suppressor T cell or Ts1 that is Ly-1+2-, functions only at the afferent limb of the anti-TMA response, binds the TMA ligand and bears cross-reactive idiotypes associated with anti-TMA antibodies. This Ts1 produces a suppressor factor (TsF1) that binds the TMA ligand, bears the cross-reactive idiotypes and I-J determinants and functions to induce an idiotype-specific Ts2 population. To study the biochemistry of this TsF, use was made of T cell hybridomas that constitutively produce TMA-TsF1 (8A.1 and 8A.3). The TsF1 was purified from culture supernatant or cell extracts by (NH4)2SO4 precipitation, reverse phase HPLC and either affinity chromatography or by preparative IEF. The TsF1 has an isoelectric point of 6.5 and a m.w. of 26,000 or 62,000 as analyzed by SDS-PAGE or high performance molecular sieve chromatography. Its precipitation in 30 to 40% (NH4)2SO4; elution pattern from reverse phase high performance columns; its capacity to bind to a mAb specific for L-glutamic acid 60L-alanine30-L-tyrosine10 (GAT)-TsF1 strongly suggest that this protein belongs to the same family of proteins as do the GAT-TsF1 described previously. Most noteworthy is that although these TsF1 proteins show remarkable similarities, they are absolutely specific in their biologic activity; TMA-TsF1 will not suppress the response to GAT-BA-TNP and GAT-TsF1 will not suppress the response to TMA-BA-TNP. Thus the TMA-TsF1 represents a second example of a unique group of Ag-specific proteins whose function is to induce or activate other suppressor T cells in the primary immune response to Ag.     

Suppressor T cell circuits in contact sensitivity. III. A monoclonal T cell hybrid-derived suppressor factor specifically suppresses local DTH transfer by a DNP-specific T cell clone

Herein we described the direct suppressive effects of a monoclonal T cell hybridoma-derived, DNP-specific suppressor T cell factor (26.10.2 TsF) on the local transfer of delayed-type hypersensitivity (DTH) by a DNP-specific BALB/c T cell clone (dD1.9). The L3T4+, Lyt-2- dD1.9 T cell clone proliferated in response to DNP-OVA and DNBS, but not TNP-OVA or TNBS, in association with I-Ed determinants present on antigen-presenting cells. Similarly, local injection of histopaque-purified dD1.9 cell blasts resulted in DNP-specific, radioresistant, I-Ed-restricted, mononuclear cell-rich ear swelling responses. Incubation in 26.10.2 TsF specifically suppressed local transfer of DNP-specific DTH by dD1.9, but not local DTH responses transferred by BALB/c T cell clones specific for TNP or GAT. The suppressive effect of 26.10.2 TsF correlated with targeting on DNP-major histocompatibility complex determinants associated with the DTH T cell (TDH) targets. 26.10.2 TsF-mediated suppression was most pronounced after exposure of dD1.9 target cells to antigen (after the stimulation phase of the T cell clone maintenance procedure), and greatly reduced when dD1.9 was cultured for long periods in the absence of DNP (after the rest phase of clone maintenance). In additional support of this hypothesis, GAT-specific TDH, normally resistant to 26.10.2 TsF-mediated suppression, were rendered susceptible to suppression after surface DNPylation. The results demonstrate a direct, antigen-specific, effector phase regulatory effect of a monoclonal TsF on a cloned, antigen-specific T cell target, and strongly suggest that suppression is mediated via targeting on DNP determinants associated with the TDH target. Simplification of complex Ts circuitry operating in suppression of the efferent limb of DTH by the use of monoclonal TsF and cloned T cell targets should provide a basis for the future study of the molecular mechanisms of immune suppression.