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CD147 or EMMPRIN belongs to the immunoglobulin superfamily of membrane receptors and is expressed in epithelial cells, cancer cells and T cells of the immune system. In T cells CD147 functions as a receptor for soluble cyclophilins and is involved in chemotaxis. We recently reported its expression to high levels and association with suppressive function in regulatory T cells. Here, we discuss its potential application as a marker of activated regulatory T cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Effector T cells have functional subpopulations with distinct cytokine, cytokine receptor, chemokine receptor and transcription factors. We review how activation of antigen specific Treg induces expression of cytokines, cytokine receptors and chemokine receptors depending upon the effector lineage they are activated by. Activated Treg express receptors that are directly related to the effector T cell lineage. Other classes of Treg are induced in the periphery from effector lineage CD4(+)CD25(-)FOXP3(-)CD127(high)T cells, either by IL-10 or TGF-β or by association with activated CD4(+)CD25(+)FOXP3(+)Treg. Thus Treg are produced and adapt to the specific immune inflammatory environment they are activated within. Activated Treg produce different molecules to mediate suppression, which are tailored to the immune response they are activated by and control., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Correction of an imbalance between allergen-specific T cell subsets is considered a critical event in establishing allergen tolerance by specific immunotherapy (SIT). In a comprehensive, longitudinal study, distinct T cell populations and Ig subtypes were analyzed in subjects allergic to birch pollen during decisive time points of SIT (i.e., induction and maintenance phase), as well as in and out of birch pollen season. An increase in Bet v 1-specific, IL-10-secreting T cells, fulfilling the criteria of inducible type 1 regulatory T (Tr1) cells, was observed by the end of the induction phase; this resulted in a decreased ratio of allergen-specific IL-5(+) Th2/Tr1 cells. In contrast, CD4(+)CD25(+)CD127(low) regulatory T cell numbers did not change. Furthermore, enhanced concentrations of allergen-specific IgG Abs were observed, whereas allergen-specific IgE and IgA levels remained unchanged. After 1 y of SIT, a reduced ratio of allergen-specific Th2/IFN-gamma(+) Th1 cells was apparent. Although untreated and SIT-treated allergic subjects developed enhanced Th2 cell responses during birch pollen season, only SIT-treated patients experienced elevated numbers of allergen-specific Tr1 cells, which were associated with reduced skin prick test reactivity and diminished clinical symptoms. In coculture assays, allergen-specific Tr1 cells showed an IL-10- and dose-dependent inhibition of CD4(+)CD25(-) T effector cells. Thus, SIT has differential effects on regulatory T cell subsets, resulting in an early induction of allergen-specific Tr1 cells associated with an increase in allergen-specific IgG, and it leads to a delayed shift from an allergen-specific Th2- to a Th1-dominated immune response.

CD8alphaalpha+CD4-TCRalphabeta+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8alphaalpha+CD4-TCRalphabeta+ regulatory T cells (CD8alphaalpha Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vbeta8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vbeta8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8alphaalpha regulatory T cells (Tregs) and found that they predominantly use the TCR Vbeta6 gene segment. In vivo injection of anti-TCR Vbeta6 mAb results in activation of the CD8alphaalpha Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8alphaalpha Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.

In patients with severe aplastic anemia (SAA), lymphocyte subpopulations were examined for the presence of HLA-DR and 2H4 (suppressor/inducer subset) antigen-expressing cells by flow cytometric analysis. Investigations were performed on peripheral blood lymphocytes before and after therapy with antithymocyte globulin (ATG) and methylprednisolone (MP), as well as on bone-marrow lymphocytes before therapy. Before treatment, only the absolute numbers of CD4+ T cells and the CD4+HLA-DR+/CD8+HLA-DR+ activated T cell ratio were significantly decreased (p less than 0.01 and p less than 0.001, respectively). Following successful ATG/MP treatment, a decrease in the CD4+/CD8+ T cell ratio was found. Regarding the suppressor/inducer subset, only absolute numbers of CD4+/2H4+ cells were somewhat higher in treated patients; the percentages were the same in all groups of patients. Studies performed on bone-marrow lymphocytes showed significantly decreased percentages of CD4+ and CD8+ T lymphocytes, which also express HLA-DR antigen. No significant changes in the distribution of activated T cells following ATG/MP therapy were found, suggesting that these cells play no major role in the pathogenesis of the disease.

Immunoregulation as a consequence of thermal injury was investigated by using a murine model involving a 30% surface area full thickness burn. Both allogeneic mixed lymphocyte reaction (MLR) and in vitro anti-SRBC responses were depressed from days 3 to 25 post-burn. Suppressor T cells could be identified in both systems between days 5 and 15. On day 5 post-burn, an Ly-1+,2-, I-J+ T cell is responsible for the majority of the suppression observed. This cell behaves like a T suppressor inducer T cell in that it must interact with an Ly-2+ cyclophosphamide-sensitive cell to manifest suppression. On day 7 post-burn, only Ly-1-,2+ suppressor T cells are found which can directly suppress the activity of Ly-1+,2- helper T cells. Thus, these cells behave as T suppressor effector cells. We suggest that feedback suppression is in operation after thermal injury, with functional suppressor inducer cells appearing on day 5 post-burn, leading to the appearance of T suppressor effector cells by 7 days post-burn. Recovery from post-burn immunosuppression occurs by day 25 post-burn and is associated with the appearance of V. villosa-adherent T cells, whose activity antagonizes that of the day 7 post-burn suppressor effector. These cells may represent contrasuppressor T cells, which could play a role in the restoration of immunocompetence after burn injury.

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.

The relative radiosensitivities of precursor (Tsp), activated (Ts) and memory (Tsm) suppressor T cells for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were investigated in mice. Spleen cells from CBA mice, primed i.v. with 10(9) SRBC 3-4 days previously, contain specific Ts cells which substantially impair the induction of DTH to SRBC in normal syngeneic recipients. Exposure of mice to 400 rad irradiation 1 day before the priming completely eliminated the subsequent development of Ts cells. In contrast, 3 days after the priming injection, Ts cell activity in mice is resistant to doses higher than 600 rads. Mice primed 40 days previously with 10(9) SRBC contain Ts-cell memory which can be readily recalled by i.p. injection of 10(8) SRBC. The secondary Ts cells which specifically inhibit DTH induction can be demonstrated adoptively in normal recipients. Mice were exposed to various doses of irradiation 40 days after the priming and 1 day before the i.p. injection. Ts memory was significantly reduced by 300 rads and was completely abrogated by 400 rads. The relative radiosensitivities of the three subsets of suppressor T cells are in the order of Tsm = Tsp greater than Ts.

A set of T cell clones (TCC) isolated from HLA-DR-, Dw-, DQ-matched allogeneic MLCs was found to proliferate autonomously when stimulated with cells carrying a wide range of class I or II specificities. This apparently unrestricted proliferation was relatively weak, and only low levels of IL-2 were present in the supernatants of stimulated cells. Autologous as well as allogeneic PBMC and B lymphoblastoid cell lines (B-LCL) were capable of stimulating such clones, which were also restimulated by suppressive, but not by helper, TCC. Moreover, such clones displayed the unusual property of autostimulation. mAb inhibition experiments suggested that class II- or class II-restricted antigens were involved in stimulation. Thus, certain "broad" mAbs (TU39, SG520) reacting with multiple locus products inhibited activation of these reagents, but none of those reacting more specifically with DR (TU34, TU37, L243, Q2/70, SG157), DQ (TU22, SPV-L3, Leu 10), or DP (B7/21), or mixtures of these mAbs, were able to do so. Evidence from sequential immunoprecipitation experiments suggested that mAb TU39 bound class II-like molecules other than DR, DQ, and DP on TCC and B-LCL, and it is therefore proposed that such putative novel class II-like molecules may carry the stimulating determinants for these autoreactive clones. DY-reactive clones lacked helper activity for B cells but mediated potent suppressive activity on T cell proliferative responses that was not restricted by the HLA type of the responding cells. Suppressive activity was induced in normal PBMC by such clones, as well as by independent suppressive clones, which was also inhibited only by mAb TU39. These findings lead to the proposal that DY-reactive autostimulatory cells may constitute a self-maintaining suppressive circuit, the level of activity of which would be regulated primarily by the availability of IL-2 in the microenvironment.

Two new monoclonal antibodies (termed 2D2 and D12) have been used to identify and to analyze phenotypically distinct subpopulations of human T cells. The 2D2 antibody recognized an antigenic determinant closely related, if not identical, to that reactive with the anti-Leu-2 monoclonal antibody. The D12 antibody reacted with a variety of cell types, which included a subpopulation of Leu-2+ (2D2+) T cells. These antibodies were used to isolate four phenotypically distinct T cell populations by sequential cell sorter techniques. Functional analyses demonstrated that the 2D2+D12+ subset was unique in its ability to suppress the antigen-induced proliferation of T cells. These cells also suppressed the proliferative responses of other T cell subsets stimulated with mitogens. Pretreatment of 2D2+D12+ T cells with mitomycin C before culture abrogated the suppressor cell activity of these cells. We propose that the cells within the Leu-2+ cytotoxic/suppressor T cell subpopulation that suppress T cell proliferation are phenotypically distinct and express the 2D2+D12+ membrane antigenic phenotype.

A single injection of TNP-labeled mouse gamma-globulin (TNP-IgG) can render the contact sensitivity response of mice resistant to suppressor cells (Tsc) and their biologically active cellfree products (TsF). Lyt-1 T cells of mice treated with TNP-IgG can protect the adoptive contact sensitivity response of immune cells from the antigen-specific suppressive effect produced by the addition of antigen-specific TsF or Tsc. When T cells of TNP-IgG-treated mice are put into culture, they produce an antigen-specific contrasuppressor factor (TcsF) that can replace the activity of the cells. When immune cells are preincubated in vitro with TcsF, they become refractory to Tsc and TsF added subsequently. The TcsF, however, has no ability to restore responsiveness to immune cells that had been previously exposed to TsF. The TcsF binds specifically to TNP, expresses an I-J-controlled determinant, and does not express standard determinants found on mouse Ig. The treatment that primes the contrasuppressor system to protect the contact sensitivity response also reportedly renders the antibody-producing system tolerant, (i.e., produces so called "split tolerance"). These results are discussed in light of the possibility that the contrasuppressor system can be responsible for so called isotype-specific immunity by rendering one arm of the immune system resistant to generalized suppressive mechanisms.

The phenotypic reconstitution of lymphoid cells in the bone marrow and peripheral blood was examined prospectively in 27 patients who underwent autologous bone marrow transplantation (ABMT) for leukemia/lymphoma. Patterns of activation within NK and T cell subsets as well as T sub-subsets were studied with monoclonal antibodies in two- and three-color FACS analysis. NK-like cells (CD16+) were found to be increased in the peripheral blood and bone marrow after ABMT. The expression of two activation markers, 4F2 and HLA-DR, was sustainedly increased within this subset. High numbers of CD4+ and CD8+ T cells carrying surface HLA-DR were found early after ABMT both in blood and marrow. The T suppressor inducer cell sub-subset (CD45R+ CD4+) was severely depressed in both the blood and marrow 6-9 months post-ABMT. Using three-color FACS analysis, half of this T sub-subset was shown to express HLA-DR+. The levels of T suppressor effector-like cells (CD11+ CD8+) remained within the normal range in both peripheral blood and bone marrow during follow-up. The HLA-DR expression was elevated and equally distributed between the CD11- CD8+ and CD11+ CD8+ sub-subset cells. There was no major impact of marrow T cell purging or CMV carrier status on the phenotypic NK/T cell reconstitution. The present results provide an immune phenotypic basis for the suggested generation of anti-leukemic NK-like and T suppressor-like activity after ABMT.

In a previous study we reported that cord blood lymphocytes show lower OKT3 responses as compared to their mothers and to other, unrelated adults. In the study reported here, we investigated the interactions between lymphocytes and adherent accessory cells in OKT3-stimulated cultures of newborn (cord), maternal, and other adult peripheral blood mononuclear leukocytes (PBML) and determined the following. (1) Removal of adherent cells (AC), by two cycles of plastic adherence or by nylon wool columns, impaired the OKT3-induced proliferation of maternal/adult cells, but significantly enhanced the OKT3 responsiveness of cord cells. (2) Addition of indomethacin, and other prostaglandin (PG) synthesis inhibitors, caused a more than twofold augmentation of cord PBML OKT3 responses, but had only a small, if any, enhancing effect on maternal/adult PBML. Cord PBML cultures deprived of AC were no longer enhanced by indomethacin. (3) Exogenous PGE2 (1.4 X 10(-6) through 1.4 X 10(-9) M) strongly inhibited OKT3-induced proliferation of maternal, cord, and adult PBML, at a wide range of antibody concentrations (5-100 ng/ml). However, an obvious difference in the extent of PG-mediated inhibition was observed among these three populations, and the order of PG sensitivity, from most to least sensitive, was cord greater than maternal greater than adult. (4) Purified interleukin-1 (IL-1) could not replace the accessory function of AC in the OKT3-induced proliferation of maternal/adult lymphocytes. In contrast, IL-1 increased by greater than 50% the OKT3 responsiveness of cord PBML in the absence, but not in the presence, of cord monocytes. Our observations strongly argue for a distinct, predominantly suppressive function of cord monocytes as compared to maternal/adult monocytes in OKT3-induced mitogenesis, and indicate prostaglandins as major mediators of this suppression.

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.

The carrier requirements for the induction of helper and suppressor T (Ts) cells were compared. Although H-2-linked Ir genes control the development of helper T cells and hapten-specific B cells, they do not influence Ts3 generation. That is, GL phi nonresponder C57BL/6 mice can generate NP-specific Ts3 cells after priming with NP-GL phi. The Ts3 cells generated under these conditions are functionally and phenotypically identical to the NP-specific Ts3 cells previously characterized. Furthermore, these Ts3 populations can be specifically depleted with a monoclonal anti-idiotope antibody prepared against monoclonal anti-NP antibodies. By using related polymers, carrier effects on Ts3 induction were noted. NP-D-GL and NP-Ficoll failed to induce Ts3 cells, whereas NP-L-GL induced this suppressor subset. The data demonstrate that Ts3 induction is independent of the carrier requirements involved in helper T cell induction and is not dependent upon B cell priming. The implications of these results with regard to the mechanisms of Ts3 induction are discussed.

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.

The present studies were carried out to characterize the cellular events involved in the induction and function of carrier-specific Ts cells, which selectively regulate the generation of IgG responses by Lyb-5- B cells. It was demonstrated that this regulation is in fact mediated by two distinct suppressor pathways. In one pathway, carrier-primed Lyt-1 + 2 - Ts cells are specifically activated by in vitro reexposure to the priming antigen. After this specific activation, these Lyt-1 + 2 - Ts cells are able to suppress IgG responses in an antigen-nonspecific manner. This suppression requires the participation of unprimed Lyt-1 - 2 + T cells, and is effective in both the early and the late phases of antibody responses. A second suppressor pathway requires the antigen-specific activation of primed Lyt-1 - 2 + Ts cells. Suppression of antibody responses by activated Lyt-1 - 2 + Ts cells is highly carrier specific, in contrast to the nonspecific effector function of Lyt-1 + 2 - Ts cells, appears to act without requirement for additional T cell populations; and is effective only early in the course of the antibody response. Thus, it appears that two Ts cell populations may function through distinct mechanisms to regulate the generation of IgG Lyb-5- B cell responses.

This study demonstrates the existence of two different suppressive systems for the regulation of antitetanus toxoid antibody production by human lymphoblastoid (LB) B cells. These B cells appear in the circulation 5 to 7 days after in vivo immunization and spontaneously secrete antibody during a 3-day in vitro culture. One suppressive system was mediated by large granular lymphocytes that exhibited high natural killer activity. This suppressive cell subset spontaneously inhibited the antibody production by autologous LB cells, and this effect could be enhanced by the addition of interferon. This inhibition of antibody synthesis could be readily reversed by the addition of as few as 10(2) K-562 cells to the culture. Additionally, the activity of this suppressive cell population could be reduced by complement (C)-mediated lysis with Leu-7 antibody. These results strongly suggest that this autologous suppression was mediated by NK cells. The other suppressor system was contained in the fraction of high density T cells depleted of Fc receptor-bearing cells, which was low in NK activity. This subset inhibited LB function in the presence of pokeweed mitogen but not interferon, and even the addition of up to 10(6) K-562 NK target cells only minimally reversed this inhibition. These results indicate that two distinct subsets of cells share regulatory functions on the in vivo induced B lymphoblastoid cells. The observation that NK cells can inhibit these highly differentiated B cells expands our view of the spectrum of natural targets recognized by NK cells.

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.

Cells from the spleen, lymph nodes, and peritoneum of DBA/2 mice bearing a subcutaneous tumor mediate nonspecific suppression of an in vitro antibody response to sheep red blood cells (SRBC) when cocultured with a normal T-cell subset(s). The spleen cells from the tumor-bearing mouse required for the suppression bear the Lyt 1 and Ala 1 surface markers characteristic of "inducer" T cells and activated cells, respectively. The activity of this cell population is also sensitive to irradiation. The normal T-cell subset which cooperates in the suppression bears the Qa-1 surface antigen which has been associated with suppressor cell precursors in several systems but lacks detectable surface Lyt 1 and 2 markers. Suppression of antibody responses in spleen cell cultures from tumor-bearing mice alone could also be elicited, but only when increased numbers of cells were cultured. These data are consistent with the theory that a tumor-activated, Lyt 1+ T-cell subset has the capacity to nonspecifically suppress immune responses by activating a Qa-1+ subset(s) of T suppressor cells, perhaps via feedback signals.

Two BSA-specific Ts cell clones have been isolated from CBA/J mice tolerized by low doses of BSA. Together with one Ts cell clone isolated from an immune animal, they have recently been characterized with regard to phenotypes and in vitro functions. In the present report the in vivo effector functions of two of them (Ts cell clones BVI/5 and HF1.MS) are described. BSA-primed lymph node cells from CBA/J mice, which had received BVI/5 or HF1.MS Ts cells at the time of immunization, do not respond to a subsequent in vitro antigenic challenge. A human gamma-globulin (HGG)-specific lymph node cell proliferation is not influenced. BVI/5 Ts cells injected into mice at the time of priming with fluorescein (Flu)-conjugated BSA or Flu-HGG inhibit the humoral anti-Flu-response in Flu-BSA-primed animals. The anti-Flu-response in Flu-HGG-primed animals is only marginally affected by BVI/5 Ts cells. The data show that it is possible to induce immunologic unresponsiveness at the humoral level by reexposing in vitro propagated Ts cell clones to their syngenic in vivo environment.

The mechanism of B cell suppression by a T cell hybridoma-derived monoclonal effector suppressor factor (TsF3) was studied in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. The NP-specific effector suppressor cells that produce TsF3 are Lyt-1-, 2+, I-J+, NP-binding T cells and are induced by immunization with NP conjugates. Monoclonal TsF3 inhibits both T cell activity as measured by suppression of contact sensitivity responses and B cell function as measured by suppression of antibody production to both T-independent and T-dependent antigens. The present studies were designed to specifically investigate the mechanisms and genetic restrictions that govern the interactions between TsF3 and its target cells in the plaque-forming cell (PFC) response. The results show that the target of TsF3 is a splenic adherent cell. Suppression will occur only if the restriction specificity of the TsF3 matches the H-2 genotype of the adherent population. Once this TsF3-adherent cell interaction has occurred, suppression of NP-specific B cells can occur across an H-2 barrier. The data also demonstrate that Igh-linked gene products do not appear to play a part in the TsF3-mediated suppression of in vitro PFC responses, which contrasts with the requirements for regulation of T cell-mediated contact sensitivity responses.

The ability of spleen cells from aged C57BL/6 mice to generate specific suppressor cells in mixed lymphocyte cultures (MLC) against allogeneic H-2 antigens was investigated. The suppressor cells from young and old mice were assayed in parallel for their ability to inhibit the proliferative response and the generation of cytotoxicity in fresh MLC. Suppressor cell generation was found to be significantly decreased in 41% of aged mice (23 to 28 mo) as compared to young controls (3-8 mo). The suppressor cells were H-2-specific, radiation-resistant (1000 R), and Thy-1+; they did not function by lysing the fresh stimulators or responder cells, or by absorbing the interleukin 2 in the fresh cultures. Suppression required very small numbers of cells to be effective. It was concluded that the effect of aging was less marked on specific suppressor cell generation than on generation of cytotoxic T cells in the MLC. However, a third type of response studied, the proliferative response, was affected earliest and most severely.

Culturing spleen cells for 2 or more days in the absence of mitogenic stimulation results in the generation of suppressor cells that can effectively inhibit the proliferative responses of freshly prepared spleen cells to mitogen or alloantigen stimulation. Suppression does not appear to be mediated by prostaglandins nor other soluble factors produced during the preculture period. The suppressor cell is described as a plastic-adherent Thy-1.2-, IgM-, FcR+ macrophage-like cell. Significant suppression of Con A responses can be detected at suppressor to target ratios as low as 1:100. The plastic-adherent suppressor is capable of terminating Con A-induced proliferation of spleen cells whether added at the onset of the Con A response or added as late as 48 hr after mitogenic stimulation. The suppressed spleen cell population displays an absence of large blast cells and a decrease in surface density of Thy-1.2 determinants.

T-cell-growth-factor (TCGF) activate peripheral blood lymphocytes (PBL), cultured for 14 days, showed killer cell activities against natural-killer resistant Daudi cells in a 4 h 51Cr-release assay. However, the effector cells obtained from patients with nonresectable carcinoma exhibited very much lower cytotoxicity to tumor cells. To analyze the mechanism of depression, we have attempted to examine suppressor cell activities of the TCGF-activated PBL. The assay for the suppressor cell activities was made by in vitro inhibition of cell-mediated cytotoxicity by incubating radiolabeled target tumor cells with lymphokine-activated killer (LAK) cells and TCGF-activated PBL. LAK cells were induced by cultivation with recombinant interleukin-2. TCGF-activated PBL, obtained from four out of ten patients with resectable carcinoma and nine out of ten patients with nonresectable carcinoma, significantly suppressed the LAK cell activities. However, this suppression was not observed in TCGF-activated PBL from ten normal healthy control subjects. TCGF-activated PBL with immunosuppressive reactivity were named lymphokine-activated suppressor (LAS) cells. To investigate the phenotypic characterization of TCGF-activated PBL, the cells were analyzed by two-color flow cytometry. TCGF preferentially expanded CD8+CD11- cells and decreased the growth of CD8+CD11+ cells in both normal healthy control subjects and gastric cancer (resectable and nonresectable) patients. Dominantly expressed CD8+CD11- cells on TCGF-activated PBL in patients--especially those with nonresectable gastric carcinoma--showed strong LAS cell activity, irrespective of the presence of killer cell activities of CD8+CD11- cells in TCGF-activated PBL from normal healthy control subjects. The results suggested the generation of CD8+CD11- LAS cells from cancer patients, and revealed that CD8+CD11- T-cells contained killer and/or suppressor cell function. In addition, it was found that the TCGF-activated PBL from gastric cancer patients were associated with an increased proportion of CD4+ Leu8+, HLA-DR+CD8+ and HLA-DR+CD25+ cells.

A mechanism responsible for the induction of NP-specific first order (inducer) suppressor cells (TS1) is described. TS1 cells are induced by i.v. administration of hapten-coupled splenic cells. Their activity is assessed by the adoptive transfer of NP-specific suppression during the afferent phase of the contact sensitivity response. NP-coupled firmly adherent, FcR+, I-A-bearing macrophages induce TS1. The antigen-presenting cells required for TS1 induction lack the Thy-1 and Lyt-1 markers, and are resistant to 500 R irradiation and to cyclophosphamide treatment. NP-coupled dendritic cells fail to induce TS1 activity. The induction of TS1 cells is genetically restricted by genes that map in the I-J region of the H-2 complex. The NP-coupled antigen-presenting cells must share at least one I-J allele with the TS1 donor for effective induction of TS1 activity. To minimize allogeneic effects in these studies, the activity of the TS1 population was assessed by adoptive transfer into syngeneic recipients. The present results are compared with the mechanisms required for the induction of second and third order suppressor cells.

Stimulation of human peripheral blood mononuclear cells (PBMC) by pokeweed mitogen bound to erythrocytes (E-PWM) has been found to result in an increased blast cell formation, lymphocyte proliferation, and enhanced immunoglobulin production, compared to stimulation of PBMC by PWM. Using flow cytometric analysis we compared the T helper/inducer (CD4+) to T suppressor/cytotoxic (CD8+) cell ratios of PBMC after stimulation by PWM and by E-PWM. E-PWM was found to induce significantly lower CD4+:CD8+ ratios on days 6 and 9 of the culture than PWM did. This effect was due predominantly to a relative increase in CD8+ T cells after stimulation of PBMC by E-PWM, compared with stimulation by PWM. However, the increase in T suppressor/cytotoxic cells on days 6 and 9 after E-PWM stimulation was accompanied by a simultaneous relative decrease in CD4+/2H4+ T cells (T suppressor-inducer cells) and by a relative decrease in CD8+/Leu-15+ T cells (CD4+/2H4+ independent T suppressor cells) on day 9, compared with stimulation by PWM. These results suggest that the greater immunoglobulin production after stimulation of PBMC by E-PWM than by PWM may be the result of a relative lack of suppression on fully differentiated B cells.

The activation of resting T cells to interleukin 2 (IL-2) production and DNA synthesis via Ti-CD3 is dependent on accessory cells (AC). Using positively selected, resting T cells activated with particle-bound anti-CD3, we investigated the ability of various cell lines to function as AC. We found that cell lines able to act as AC all expressed LFA-3, while cell lines not expressing LFA-3 were unable to provide AC signals. This applied to CD3+, CD4+, and CD8+ T cells. Sheep red blood cells (SRBC), which express LFA-3-like molecules, also had a weak, but significant AC function in this test system. Both CD4+ and CD8+ T cells activated with particle-bound anti-CD3 could be induced to enter DNA synthesis in the absence of AC when monoclonal antibodies reacting with CD2 were present instead of AC. IL-2 production could be detected in the latter cultures but not when positively selected CD3+ or CD2+ T cells were cultured alone. Our data suggest that activation of resting T cells via CD3 will lead to IL-2 receptor expression, while the interactions between LFA-3 and its ligand CD2 provide the necessary secondary signals for IL-2 production and induction of DNA synthesis.

Two suppressor cell populations that interact to down-regulate in vivo development of the cytolytic T-cell (CTL) response to trinitrophenyl-modified syngeneic spleen cells (TNP-SC) have been further characterized. Suppressor cells induced by the iv injection of trinitrophenyl-modified syngeneic spleen cells possess Thy 1.2 antigen. Their precursors are insensitive to pretreatment of host animals with cyclophosphamide (CY). Suppressor cells that arise after dermal sensitization with trinitrochlorobenzene are also Thy 1.2 antigen positive but their precursors are sensitive to pretreatment with CY. These characteristics of the two suppressor T cells (Ts) are identical to those of the two Ts that are generated by similar methodologies and that together suppress contact sensitivity (CS) to picryl chloride. Neither the CS nor CTL response was suppressed when host animals possessed only one set of Ts. In contrast to suppression of CS at the efferent phase, development of CTL was suppressed only when the two Ts were present early during sensitization (afferent phase). Since the results point to several similarities between the two sets of Ts that are active in the down-regulation of the CS and CTL responses, it is suggested that the two dissimilar immune responses directed to the same hapten, namely CS and CTL, may be controlled by the same suppressor cells. Since it appears that the two sets of Ts interact to affect different phases of the CS and CTL responses, down-regulation of each must be accomplished through different mechanisms.

Spleen cells from 3 different strains of mice (C57 (H-2b), CBA (H-2k) and BALB/c (H-2d] were stimulated in vitro with different concentrations of concanavalin A (CA) for 48 h. This resulted in the production of cells capable of inhibiting the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) in a mixed lymphocyte culture (MLC). 1 microgram/ml was an effective concentration of CA to induce C57 and BALB/c suppressor cells (SC), but 5 micrograms/ml CA was required to induce CBA SC. SC precursors (SC-P) were shown to be radiosensitive and the results suggest that SC themselves may be radiosensitive. SC were effective in the presence of added interleukin-2 (IL-2). SC were then induced at limit dilution in microwells in a volume of 25 microliter. A MIC (200 microliter) was then (after 48 h) added to each microwell. This resulted in a dilution of the concentration of CA to a level below which it was effective at inducing suppression. Cytotoxicity was then assessed 7 days later. It was thus possible to analyse SC-P at the clonal level and estimate their frequency. The frequency of C57 splenic SC-P (active against a C57 anti-BALB/c MLC) was 14.4 X 10(-6), the frequency of CBA splenic SC-P (active against a CBA anti-BALB/c MLC) was 92.3 X 10(-6), and the frequency of BALB/c splenic SC-P (active against a BALB/c anti-CBA MLC) was 15.8 X 10(-6). It was possible to analyse SC-P at a clonal level whether or not the MLC contained added IL-2. SC and SC-P were shown to be sensitive to anti-Thy-1 and complement.

The present longitudinal study was designed to characterize immunosuppression during acute Plasmodium falciparum infection, during the treatment and up to 1 month after the acute stage. The proliferative responses of blood mononuclear cells (BMNC) isolated from non-immune and semi-immune malaria patients and controls to mitogens and two Plasmodium-derived stimulators (merozoites, Meroz, and soluble purified antigen, SPag) and non-related antigens were measured by [3H]thymidine incorporation. BMNC isolated before treatment (day 0) from the non-immune patients did not respond to Meroz, whereas those from controls showed a significantly higher response. The SPag responses were also low in BMNC isolated on day 0 and increased in both the non-immune and the semi-immune patients during the observation period. These findings indicate that during malaria there is a depression of the parasite-specific proliferative response. The subset composition of BMNC isolated from non-immune patients was studied in a FACS analyser. The mean cell volumes of both Leu 2+ and Leu 3+ cells were increased during the acute phase of the infection, indicating that malaria infection results in activation of both T-helper and T-suppressor cells. There was no overall reduction of the response to mitogens on day 0. However, 3 days after initiation of the treatment the mitogen response was decreased. This finding indicates that it is important to distinguish between the effects of malaria infection and of drug treatment.

The ability of UV-treated splenic adherent cells (SAC) to induce T cell-mediated immunity and suppressor T cells was analyzed in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. UV irradiation of 0.88 KJ/m2 decreased the capacity of NP-coupled SAC to induce delayed-type hypersensitivity (DTH) responses by about 50%. The ability of uncoupled UV-treated SAC to induce allogeneic DTH response was also imparied, indicating that UV-treated SAC are inefficient at inducing DTH in these systems. TS1 induction by UV-treated NP-SAC was evaluated TS1 induction by UV-treated NP-SAC was evaluated by using adherent cells that were subjected to the same dose of UV irradiation that impaired DTH induction. Intravenous administration of 10(3) or 10(4) UV-treated NP-coupled SAC induced TS1 cells with the same efficiency as non-UV-irradiated cells. The TS1 cells induced in this fashion were antigen specific. Furthermore, to establish that the antigen was not reprocessed by the host, I-J-mismatched, UV-treated NP-SAC were unable to induce TS1 cells. The population of antigen-presenting cells responsible for TS1 induction appear to express both I-A and I-J determinants. TS2 induction by UV-treated accessory cells was also analyzed. TSF1 inducer suppressor factor was pulsed onto graded numbers of either normal or UV-treated adherent cells. The same levels of antigen-specific suppression were induced with normal and UV-treated cells. Finally, TS3 induction by UV-treated NP-SAC was analyzed. UV-treated and normal NP-SAC (3 X 10(3] induced antigen-specific suppression of NP DTH responses. I-J-mismatched, UV-treated NP-SAC failed to induce suppression, suggesting that the hapten was not reprocessed by the host under these experimental conditions. The accessory cell population responsible for TS3 induction appears to express both I-A and I-J determinants. Thus, there are at least two functional distinctions between the antigen-presenting cells that induce immunity vs those that induce suppressor cells. First, UV treatment selectively impairs the antigen-presenting cells, which activate the positive limb of the immune response. Second, I-J determinants appear to be specifically associated with the SAC, which induce suppressor T cells. Although these criteria can be used to distinguish the accessory cells involved in suppressor cell pathways from those controlling helper T cell induction, there were no discernible phenotypic differences among the accessory cells that induce the TS1, TS2, and TS3 subsets.

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.

The ability of antigen-coupled splenic adherent cells to induce suppressor T cells (Ts) is dependent on the presence of I-J determinants on antigen-presenting cells. After 4 days of in vitro culture, antigen-coupled adherent cells lose the capacity to induce Ts. Supernatants from Con A-stimulated lymphocyte cultures and purified interferon-gamma can sustain accessory function for the induction of Ts. Furthermore, after in vitro culture of splenic adherent cells, there is an apparent correlation between the loss of I-A determinants and the decrease in I-J-restricted Ts induction. Stimulation of Ia expression with interferon-gamma results in a simultaneous increase in the ability to induce Ts. Finally, elimination of I-A-bearing splenic adherent cells with antibody + C eliminates I-J-restricted Ts induction. The combined data imply a co-regulation of I-A and I-J on the antigen-presenting cells involved in the induction of both the Ts1 and Ts3 suppressor T cell subsets.

Immunogenic stimuli rapidly induce a potent mediator with antisuppressor activity which represents complexes of Ig and antigen. The formation of the complexes depends on the interaction of two T cells both of which bear the Ly1 phenotype. The two T cells can be separated on the basis of their sensitivity to antilymphocytic serum and dependency on the presence of thymus. T cells bearing I region coded determinants are essential for the formation of the mediator.

Following activation with concanavalin A (Con A), murine T cells are able to suppress the generation of allospecific cytotoxic T lymphocytes (CTL). We have analysed the phenotype, tissue distribution, and mode of action of these cells in an effort to understand further the regulation of CTL-mediated immunity. The precursors of such cells are rare (1 cell per 70,000 spleen cells being able to suppress the generation of a particular allospecific response), but are much more abundant in the spleen than in the thymus. By the use of cytotoxic antibodies, we have been able to demonstrate that the splenic precursors of such cells are Thy-1.2+, CD4-, CD8- but, following activation with Con A, these cells acquire the CD8 marker. Cellular suppression by these lymphocytes is dramatically increased in the presence of the Th2-derived lymphokine, IL-4, whereas IL-2, the Th1-derived lymphokine, significantly augments the generation of CTL in a mixed lymphocyte culture even though relative suppression is still evident in the presence of Con A-activated lymphocytes. Suppression is not due to overcrowding of a cell culture since adding Con A-activated cells to an A anti-B + C culture often resulted in the suppression of the A anti-B response but not the A anti-C response, or vice versa. Suppression appears to require cellular interaction since supernatants from Con A-activated lymphocytes are unable to mediate suppression. Such cells may play an important intermediate role in homeostasis.

15-hydroperoxyeicosetetraenoic acid (15-HPETE), a lipoxygenase metabolite of arachidonic acid, inhibited polyclonal IgG and IgM production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood mononuclear cells, whereas 15-hydroxyeicosetetraenoic acid (15-HETE) had little effect in this system. T cells preincubated for 18 hr with 15-HPETE caused substantial inhibition of IgG and IgM production of fresh, autologous B and T cells stimulated by PWM. The suppressive effect of the 15-HPETE-treated cells was lost if the cells were irradiated before the PWM culture, but not by treatment with mitomycin C. The suppressive effect was also lost if OKT8+ T cells were removed after, but not before, preincubation of the T cells with 15-HPETE. OKT8- T cells incubated with 15-HPETE for 18 hr showed a large increase in the percentage of cells staining with directly fluoresceinated Leu-2, another marker for suppressor cells. Thus, 15-HPETE induces functional and phenotypic suppressor cells from resting human peripheral blood T cells.

Idiotope-specific immunoenhancement or suppression was induced in C57BL/6 mice by the injection of physiological amounts (100 ng-10 micrograms) of monoclonal anti-idiotope antibody. As previously described, nanogram doses enhanced idiotope expression while a 10-micrograms dose of anti-idiotope antibody induced the activation of a population of Thy 1.2+, Lyt 1-, 2+ suppressors. Both positive and negative regulatory activities were confined to the non-mu, idiotope+ compartment of the plaque-forming cell response. Administration of intermediate doses of anti-idiotope antibody resulted in an immune state indistinguishable from that of naive mice. This apparently normal response was in fact the product of a simultaneous activation of balanced enhancing and suppressive activities. When treated with anti-Lyt 2 or Lyt 1 and complement, spleen cell populations taken from such phenotypically "naive" mice revealed latent idiotope-specific immunoenhancement or suppression, demonstrating the components of a functional regulatory equilibrium.

Previous studies demonstrated that patients with active systemic lupus erythematosus (SLE), especially those with active renal disease, had a marked reduction in T4+2H4+ suppressor inducer cells in their peripheral blood. However, it was puzzling to find that active SLE patients without renal diseases often had normal percentages of T4+2H4+ cells. In the present study, we attempted to determine whether active SLE patients bearing normal percentages of T4+2H4+ cells had a defect in their expression of the 2H4 molecule on T4+ cells after autologous mixed lymphocyte reaction (AMLR) activation. The peripheral blood lymphocytes (PBL) from 50 SLE patients with normal percentages of T4+2H4+ cells (greater than or equal to 7% in PBL) were studied and the results were compared with those of 40 normal individuals. The density of the 2H4 molecule on T4 cells from normal controls increased during the 7-d AMLR; in contrast T4 cells from patients with SLE, especially those with active SLE, had defective expression of the 2H4 antigen after AMLR activation. Patients with inactive SLE, like normals, showed an increase in the 2H4 molecule after AMLR activation. Moreover, a strong correlation was observed between percent suppression of pokeweed mitogen (PWM)-driven IgG synthesis and the density of the 2H4 antigen on AMLR-activated T4 cells. Serial analysis of patients with SLE showed that the density of the 2H4 antigen expression and the suppressor inducer activity of AMLR-activated T4 cells were inversely correlated with disease activity. Thus, defective expression of the 2H4 antigen may be an important mechanism for the failure of active SLE patients with normal percentages of T4+2H4+ cells to generate suppression.

Infection with Plasmodium falciparum induces marked disturbances in normal immunoregulatory functions. Antigen-specific immunosuppression is a feature of acute malaria and has been linked to activation of CD8+ T suppressor cells. Among immune adults, cell-mediated immune responses to malaria antigens are extremely variable when measured in vitro, and there is no obvious relation between responsiveness and resistance to clinical disease. In this study, when CD8+ cells were removed from peripheral blood mononuclear cell preparations obtained from individuals who responded poorly to a soluble malaria antigen preparation, both lymphoproliferation and gamma interferon production were significantly enhanced, but responses to other soluble antigens and mitogen were unaffected. No effect of CD8+ cell depletion was seen in individuals whose undepleted mononuclear cells gave a high response to the malaria antigen. This suggests that for some malaria-exposed individuals, CD8+ cells activated in vitro by exposure to malaria antigens suppress other cellular responses and may obscure the presence of potentially protective immune mechanisms.

Leukotriene B4 (LTB4) can regulate several lymphocyte functions, including the augmentation of cytotoxic activity and the induction of suppressor cells. When T lymphocytes were preincubated with picomolar concentrations of LTB4, they would suppress the proliferative response of unfractionated peripheral blood mononuclear leukocytes to concanavalin A in a subsequent co-culture system. Such a suppression did not occur when the responding population was depleted of monocytes. Furthermore, the effect was reversed to an enhancement when the responding unfractionated population was treated with indomethacin, suggesting a role for monocytes and cyclooxygenase products in the effector phase of LTB4-induced suppressor activity. When sorted into T4+ and T8+ cells before preincubation with LTB4, both T cell subsets could be induced by LTB4 to exert suppression. T4+ cells, however, required the presence of monocytes in the responder population in order to manifest suppressor activity, whereas T8+ cells were active even in the absence of monocytes. When LTB4-preincubated T cells or T4+ cells were sorted into T4+ and T8+ subsets after preincubation, suppressor cell activity was found only in the T8+ subset. Furthermore, T8+ cell-depleted T lymphocyte cultures, incubated for 24 hr with LTB4, showed a significant increase in the proportion of T8+ cells. Together, these data suggest that LTB4 induces suppressor T cells which can derive from either T4+ or T8+ subpopulations but which are phenotypically T8+ when exerting their suppressive activity. Thus, by interacting with both T4+ and T8+ lymphocytes, LTB4 can modulate immune responses with the cooperation of functionally competent accessory monocytes.

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.

We previously demonstrated that immunization with low (10 micrograms) doses of high m.w. polysaccharide from the gram-negative bacterium Pseudomonas aeruginosa generates T cells that suppress the ability of antibacterial T cells (Tab) to protect against bacterial infection. The current studies indicate that Ts cells with properties identical to those elicited by low dose polysaccharide immunization can be generated by immunization with Tab. Tab-elicited Ts cells can abrogate in vivo induction and in vitro and in vivo expression of antibacterial T cell activity. Tab-elicited Ts are Ag-specific and H-2 restricted in their suppressor activity. Non-immune T cells fail to elicit suppressor activity. These studies provide additional evidence that the protective T cell response to P. aeruginosa is controlled by a network of T cells that are probably recognizing idiotypic determinants on P. aeruginosa-immune T and B cells.

Alloantigen-specific suppressor T cells are activated from normal murine spleen cells in mixed lymphocyte reactions (MLR). These T cells are radioresistant and suppress the activation of cytotoxic T lymphocytes (CTL) in second primary MLR cultures. This report demonstrates that cyclosporin A (CsA) blocks the activation of these suppressor cells at a dose of 1 microgram/ml. However, reconstitution of CsA blocked cultures with IL 2 restores the activation of the suppressor T cells, but fails to significantly restore the activation of CTL in these same cultures. This differential activation requirement was used to establish T cell lines that demonstrate enriched suppressor cell activity but depletion of CTL activity. These findings are discussed in terms of the mechanism of action of CsA in these distinct T cell subsets and the relevance to models of allograft unresponsiveness.

We have examined the characteristics of a rat suppressor T cell (Ts) that inhibited the antigen-dependent proliferative response of antigen-primed T cells. The kinetics of in vitro induction of Ts from lymph node T cells obtained from antigen-primed rats indicated that Ts were induced in the presence of the priming antigen within 48 hr of culturing. The Ts produced during the first 48 hr of in vitro cultures were radiosensitive (2000 rad) but became partially radioresistant within the next 48 hr of culturing. In the presence but not the absence of priming antigen, Ts inhibited the antigen-dependent proliferative response to the priming antigen as well as to heterologous antigens. Suppression appeared to be mediated via a nondialyzable suppressor factor (TsF). The induction of Ts in cultures required the presence of OX-6-/OX-8- T cells, antigen-presenting cells, and the antigen. Although a majority of cells recovered from the induced cultures were OX-8+, there was no evidence that OX-8+ antigen expression per se was related to Ts activity. Addition of highly purified IL 2 augmented the Ts-mediated suppression. The immunoregulatory implications of these findings are discussed.

Suppressor T cells (Ts-1) induced with trinitrophenyl (TNP)-conjugated syngeneic spleen cells (TNP-SC) can be enriched on antigen-coated plates and are afferent suppressors. In addition, these suppressor cells produced soluble suppressor factors (TsF) that were active in vivo. Therefore, the Ts-1 cells in the TNP system are very similar to the Ts-1 cells in other systems we have studied earlier. Further characterization of these TsF-1 revealed that TsF-1 obtained from TNP-SC-induced Ts-1 is major histocompatibility complex restricted in its activity. Injection of TNP-specific TsF-1 into naive mice did not induce Ts-2 unless additional corresponding antigen was provided. Moreover, the Ts-2 cells induced by administration of both TsF-1 and trinitrobenzene sulfonic acid were antigen specific rather than antiidiotypic.

Nonspecific suppressor cell (SPC) activity has been induced in vitro by preculturing splenocytes from normal mice in the presence of mouse amniotic fluid (MAF) and alpha-fetoprotein for 5 days or more. In adoptive transfer experiments in vivo, these AFP-precultured SPC were shown to reduce the humoral response of mice to sheep red blood cells and the cell-mediated cytotoxic response to allogeneic tumor cells. In mixing experiments in vitro, using freshly explanted splenocytes, the AFP-precultured splenocytes abrogated the generation of specific cytotoxic T-lymphocytes in primary mixed lymphocyte-tumor cell cultures. Supernatants of such precultured cells had at best only a marginal effect. These suppressor cells were found to be nylon-wool nonadherent and their effect could be almost completely abolished by treatment with anti-Thy-1, 2 serum plus complement. SPC precursors were found to be sensitive to cyclophosphamide (in vivo) and to hydrocortisone (in vivo and in vitro). At the same time, they are resistant to different doses of radiation.

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.

In the 4-hydroxy-3-nitrophenyl acetyl (NP) suppressor system, third order suppressor cells (Ts3) subset of suppressor cells is generated after Ag priming, but, in order to express suppressor activity, these cells need to be further activated or triggered with a specific second order suppressor factor. By in vitro activation of Ts3-containing lymph node cells or a pTs3 hybridoma we now show that macrophages are also required for Ts3 activation. In addition, we demonstrate that IJ genetic restrictions control this activation process. Furthermore, we directly demonstrate Ts3 activation using cloned macrophage hybridoma cells. To further investigate the interactions between Ts3 cells and the accessory cells involved in their activation, we attempted to block the second order suppressor factor mediated activation of Ts3 cells with antibodies. The activation of Ts3 cells can be blocked by the addition of anti-IJ, anti-IJ idiotype or anti-NPb idiotype antibodies, but not by anti-CD8, anti-IA, or anti-IE antibodies. Anti-IJ mAb blocked Ts3 activation at the lymphocyte level whereas anti-IJ idiotype blocked activation at the accessory cell level. Finally we tested, whether these antibodies can also directly activate primed Ts3 cells. We demonstrate that cross-linked anti-IJ, anti-NPb and anti-CD3 antibodies can activate Ts3 cells. The results are discussed in terms of receptor-ligand structures on Ts and accessory cells which are required for the activation of Ts3 cells.