The immune response and the eye. II. The nature of T suppressor cell induction in anterior chamber-associated immune deviation (ACAID)

We studied the cellular basis for the induction of Ts cells in anterior chamber (AC)-associated immune deviation (ACAID) by using TNP-modified syngeneic spleen cells (TNP-Spl). We demonstrate that the cells responsible for the induction of TNP-ACAID are non adherent, IA- T cells. This is in contrast to the antigen-presenting cells which induce suppression after the i.v. injection of TNP-Spl which are IA+/I-J+ adherent cells. Furthermore, two T cells within the TNP-Spl population are required to initiate suppression in TNP-ACAID: one is Lyt-1+, and I-J+, the other is Lyt-1+ and reactive with a monoclonal antibody, 14-30, which specifically identifies Ts inducer cells. The antigen specificity of ACAID resides in the 14-30+ T cell, and not the I-J+ cell. Although both cells must be viable to induce suppression, neither they (nor their products) must be in direct contact within the eye; one population may be in the right AC, the other in the left. Our results suggest that it is Ts inducer cells placed into the AC of the eye which initiate TNP-ACAID, and that these cells exit (or secrete Ts factors which exit) the eye to induce Ts effector cells in the spleen.     

Idiotype-specific T suppressor factor alternatively interacts with a nonspecific T-acceptor-like cell to mediate immune suppression

The ability of the idiotype (Id)-specific second-order T suppressor factor (TsF2) to interact with a final effector Ts cell type other than the previously reported third-order Ts (Ts3) subset was studied in the phenyltrimethylamino (TMA) hapten system. Hence, mice were primed with unrelated heterologous haptens to induce the nonspecific T acceptor (Tacc) cells following published procedures. When enriched T cell populations containing these nonspecific Ts were briefly incubated in vitro with TMA-TsF2, they produced suppression upon adoptive transfer into cyclophosphamide-treated mice which had been previously immunized for TMA-specific delayed-type hypersensitivity. Despite the fact that the effector population studied in this report also required Id-binding TsF2 for its function, it differs markedly from the Ts3 subset studied previously in the TMA system. First, the cell type studied herein could be easily generated with noncrossreacting heterologous chemically reactive haptens when applied directly to the skin of mice. Furthermore, these Ts effector cells had no detectable intrinsic receptors for homologous haptens and most importantly, unlike Ts3, this population had no affinity for the TMA hapten. Nevertheless, the nonspecifically induced Ts once activated by TsF2 suppresses TMA-directed, but not similar immune responses specific for heterologous haptens. Thus the results indicate that TsF2 can functionally interact with a final effector Ts subset (very similar to the Tacc) other than the well described Ts3 population. The ramifications of these findings are discussed with reference to a generalized view of the cellular basis of terminal phases of immune suppression.     

Regulation of the systemic immune response by visible light and the eye

The injection of certain antigens into the anterior chamber (AC) of the eye results in the induction of antigen-specific suppressor T cells (Ts cells), which inhibit systemic delayed-type hypersensitivity (DTH). We have previously shown that down-regulation by Ts cells after AC injection with 2,4,6-trinitrophenol (TNP)-coupled spleen cells (TNP-Spl) is initiated by the intraocular activation of Ts inducer cells. These cells activate T suppressor-effector cells in the spleen that are responsible for suppressed DTH. With dark- and light-reared mice (Balb/c), we show that visible light has a direct effect on the intraocular T cell reaction that leads to systemic suppression. Our results show that if light is prevented from reaching the eye by dark rearing, by placing light-reared animals in the dark after AC injection, or by closing the eyelids of light-reared animals after AC injection, Ts cells are not activated. We show that light is responsible for establishing conditions in the eye that cause the preferential activation of Ts cells. The intraocular conditions established by light are not developmentally mandated as is visual development, but can be eliminated in adult light-reared animals by placing them in the dark for 18 h after AC injection. These conditions can also be induced in adult dark-reared animals by returning them to the light for just over 24 h before AC injection. These studies have important implications for understanding intraocular immune responses and possibly for the treatment of eye disease.     

Accessory cells in immune suppression. III. Evidence for two distinct accessory cell-dependent mechanisms of T lymphocyte-mediated suppression

Suppression of antibody secretion by the 2,4,6-trinitrophenol (TNP)-binding BALB/c myeloma, MOPC 315, by idiotype- and hapten-reactive suppressor T cells is mediated by secreted factors (TsF) and requires the presence of accessory cells (AC). Idiotype-specific TsF functions only in the presence of Ia+ AC and is completely idiotype specific. Moreover, no suppression is observed when myeloma targets and AC are separated by cell-impermeable membranes, indicating that the role of AC may be to bind, focus, and/or present TsF to the myeloma cells. In contrast, TNP-specific TsF inhibits myeloma function in the presence of TNP-protein and activated macrophages that are not Ia+. This form of suppression is nonspecific at the effector stage; i.e., anti-TNP TsF inhibits a non-TNP binding cell line, TEPC 15, as long as TNP-protein and activated macrophages are present. Moreover, suppression occurs even when myeloma targets and AC are separated by cell-impermeable membranes. These results are consistent with the view that hapten-reactive TsF binds to antigen on the surface of macrophages and induces these cells to secrete nonspecific immunosuppressive molecules. Thus, different types of AC may play fundamentally different roles in TsF-mediated suppression; they may either bind and present TsF to targets (as in the case of idiotype-specific TsF) or secrete nonspecific immunosuppressants as a consequence of a TsF-antigen interaction (hapten-specific TsF). Autonomous, suppressible targets provide valuable experimental systems for analyzing the cellular interactions in T cell-mediated suppression.     

The immune response and the eye. III. Anterior chamber-associated immune deviation can be adoptively transferred by serum

After the anterior chamber (AC) injection of trinitrophenol-coupled (TNP) spleen cells, it is observed that systemic delayed-type hypersensitivity responses to TNP are inhibited by Ag-specific suppressor T cells. We recently reported that suppression is initiated by viable TNP-coupled T cells within the inoculum and upon further analysis we found that these cells have the surface phenotype of CD4+ Ts inducer cells. We report here that treatment of these TNP-T cells with cycloheximide or cytochalasin-B before to AC injection abolishes suppression, whereas treatment with 2000 rad radiation does not. This indicates that protein synthesis and secretion are required to initiate suppression but proliferation is not. Further, we demonstrate the adoptive transfer of suppression by serum of AC inoculated animals. Detection of the component in serum in adoptive transfer assays, however, requires removal of the spleen before AC injection. We establish that the material in serum is a Ts cell product (T suppressor-inducer factor) based on three criteria: it is Ag specific, genetically restricted, and reactive with a mAb that specifically identifies these molecules. These results suggest that the signal leaving the eye to induce suppression of delayed-type hypersensitivity is T cell derived and that molecules mediating immune regulation for this organ are made within the eye and transported via the serum to the spleen.     

Regulation of the immune response to tumor antigens. X. Activation of third-order suppressor T cells that abrogate anti-tumor immune responses

The experiments described further define the suppressor T cell pathway in the S1509a tumor system. We demonstrated previously that S1509a-induced Ts1, TsF1, and Ts2 specifically suppress in vivo Ly1+2- T cell-dependent responses to S1509a and that Ts1 suppress in vivo Ly-1+2- T cell-mediated proliferative responses to S1509a. We have now shown that in vivo administration of either S1509a-induced TsF1 or TsF2 suppresses both in vivo and in vitro Ly-1+2- T cell-mediated responses to S1509a. Furthermore, we revealed the existence of Ts3, which are activated by S1509a tumor antigen and TsF2, in this murine tumor system. Finally, we demonstrated that cyclophosphamide abrogates the suppressive effect of TsF2 but not that of Ts3. These results are discussed with respect to T cell-mediated suppression in other murine tumor systems and the possible pivotal role for a tumor antigen-presenting cell in activating Ts3 in the S1509a tumor system.     

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.     

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.     

Infectious and noninfectious tolerance are blocked by a monoclonal antibody to T-suppressor factor

Two forms of hapten-specific unresponsiveness have been demonstrated following intravenous (iv) injection of hapten-conjugated syngeneic spleen cell based on the nature of the antigen-presenting cell (APC): I-J+, I-A- APC have been shown to induce T-suppressor cells (Ts cells) which are demonstrated upon adoptive transfer, while I-J-, I-A+ APC induce a nontransferable tolerance. In this paper we report that a monoclonal antibody specific for T-suppressor effector cells and factors (14-12) can block the Ts cells induced by I-J+, I-A- APCs and the tolerance induced by I-J-, I-A+ APCs. In addition, it sufficiently overcomes suppression such that injection of TNP-spl iv induces immunity rather than suppression. We show that the I-A+, I-J- TNP-spl, which induce nontransferable tolerance upon iv injection, are the cells which induce immunity in 14-12-treated recipients. These results demonstrate that injection of I-J-, I-A+ APC does not lead to clonal deletion and the tolerance induced by the iv injection of both I-J+, I-A- and I-J-, I-A+ APC operate via Ts cells.     

Age-related changes within a suppressor T cell circuit

The effects of aging on cellular and molecular components of the 4-hydroxy-3-nitrophenyl acetyl-specific suppressor T (Ts) cell circuit were analyzed in vitro using inducer (Ts1), transducer (Ts2), and effector (Ts3) cells and activating factors (TsF1 and TsF2) derived from young or old mice. The activation of Ts2 cells by TsF1 and of Ts3 cells by TsF2 was found age-restricted, suggesting a loss of Ts2 and Ts3 cell subsets in old mice. However, the activation of Ts3 cells by small amounts of TsF2 is more efficient when both are derived from old rather than from young mice while the same level of maximum suppression is attained. Higher affinity of the interactions involved in Ts cell activation may compensate for loss of Ts cell subsets in old mice. No age restriction was found for antigen presentation to Ts1 cells and for the interaction between Ts3 cells and target B cells. Thus, the effects of aging on immunosuppression result from changes within the Ts cell circuit.     

Type II Collagen Induces Peripheral Tolerance in BALB/c Mice via the Generation of CD8+ T Regulatory Cells

Antigens introduced into the anterior chamber (AC) of the eye induce a potent form of antigen-specific peripheral immune tolerance termed AC-associated immune deviation (ACAID), which prevents inflammatory immune responses and is characterized by impaired delayed-type hypersensitivity (DTH) responses. Type-II collagen (CII) is a fibrillar protein expressed exclusively in cartilage tissues. Although of its clinical relevance to Rheumatoid arthritis, aging, and osteoarthritis, there have been no studies to date to test if CII has the ability to induce ACAID. We hypothesized that ACAID could be generated via AC injection of CII in BALB/c mice. Using a DTH assay, the hypothesis was supported and led to another hypothesis that CII is capable of inducing specific immune tolerance via CD8⁺ T regulatory cells (Tregs). Thus, we performed functional local adoptive transfer (LAT) assays to examine the regulatory roles of spleen cells, T cells, and CD8⁺ T cells in the specific immune regulation induced by CII injection into the AC. Results indicated that CII induced ACAID when injected into the AC. Spleen cells of mice injected with CII in the AC significantly suppressed DTH responses. The T cell compartment of the spleen was capable of expressing this suppression. CD8⁺ Tregs could solely express this CII-driven suppression and even exerted more noticeable suppression than spleen cells or splenic T cells. This study suggests a crucial role for CD8⁺ Tregs in mediating CII-driven ACAID-mediated immune tolerance. This could have therapeutic implications in Rheumatoid arthritis, aging, osteoarthritis, and other diseases in which CII is involved.     

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.     

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.     

Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice.

A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE.     

Induction of anterior chamber-associated immune deviation (ACAID) by allogeneic intraocular tumors does not require splenic metastases

Anterior chamber-associated immune deviation (ACAID), induced by intracameral injection of allogeneic tumor cells, is expressed in three distinct ways: 1) progressive growth of intraocular tumors, 2) specific suppression of systemic allograft immunity, and 3) transient growth of allogeneic tumors injected subcutaneously. Induction of ACAID requires that alloantigen presentation occur via the anterior chamber; injection by other routes failed to elicit this phenomenon. Antigenic material must remain in the anatomically intact eye for at least 10 days; removal of the injected dye before this time prevented the establishment of ACAID. The similar temporal requirement for an anatomically intact spleen confirms the validity of the concept of a camero-splenic axis for processing of intracamerally injected alloantigens. Deployment of an alternate model of ACAID, using LP/J mice injected intracamerally with B16F10 melanoma, showed the antigen-specific inductive signal for ACAID (transmitted via the camero-splenic axis) was not in the form of viable alloantigen-bearing tumor cells that metastasize to the spleen. B16F10 melanoma cells were never found in the spleens or any other extraocular sites after intracameral injection, despite the fact these mice manifested ACAID and harbored enormous ocular tumors. The data emphasize that intraocular processing of antigens is a unique and dynamic phenomenon with significant, systemic immunologic consequences.     

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)     

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.     

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.     

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.