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

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

Modulation of suppressor T cell induction with gamma-interferon

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.     

Distinct populations of antigen-presenting cells are required for activation of suppressor and contrasuppressor T cells by type III pneumococcal polysaccharide

Type III pneumococcal polysaccharide (S3) coupled to spleen cells (S3-SC) has been shown to activate S3-specific Ts and Tcs in mice. Ts activation required I-J identity between carrier SC and Ts donors whereas I-A identity was required for Tcs activation. The carrier SC therefore presumably function as APC for Ts and Tcs activation by S3 since they are apparently not represented by APC present in the Ts and Tcs donors. The properties of the APC required for activation of S3-specific Ts and Tcs were determined by coupling S3 to various spleen cell subpopulations and assessing the ability of the various S3-SC populations to activate Ts and Tcs. The results indicate that Ts and Tcs are preferentially activated when S3 is presented on distinct cell types. S3-specific Ts were activated when S3 was coupled to plastic adherent cells. These cells are nonadherent to anti-Ig and nonfunctional in cyclophosphamide (Cy)-treated mice and their function is eliminated following treatment of cells with either anti-I-A or anti-I-J and C. In contrast, S3-specific Tcs were activated when S3 was coupled to anti-Ig adherent SC which bear I-A and the B cell marker J11d. These cells are functional in Cy-treated mice and their function is resistant to treatment with anti-I-J and C. Thus presentation of S3 on distinct cell types results in the preferential activation of T cells having opposing immunoregulatory function.     

A role for macrophages in suppressor cell induction

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.     

Splenic I-J-bearing antigen-presenting cells in activation of suppression: further characterization

A set of I-J-bearing murine splenic antigen-presenting cells (APC) has been found to be responsible for first order suppressor cell (Ts1, afferent suppressor cell) activation in the azobenzenearsonate (ABA) hapten system after intravenous administration. Suppressor cells induced by this set of hapten-coupled cells do not function in the efferent phase of the delayed hypersensitivity (DTH) response. The functional activity of this novel APC to activate afferent suppressor cells was resistant to a dose of ultraviolet radiation (UVR) sufficient to largely abrogate the ability of splenic APC to immunize for a DTH response. It was also found that the previously described splenic I-J-bearing APC needed for third-order suppressor cell (Ts3, effector-suppressor cell) activation is adherent and UVR resistant. The sets of I-J-bearing APC appear to be crucial elements in the activation of suppression and thus in determining the balance between immunologic reactivity and unresponsiveness. Furthermore, the UVR resistance of this set of novel APC may be relevant to the in vivo effects of UVR exposure to mice.     

The differential ability of splenic adherent cells from B6.lpr mice to induce suppressor T cells

Hapten-coupled splenic adherent cells or resident peritoneal cells from autoimmune B6.lpr mice that are over 5 mo of age fail to induce first-order inducer suppressor T cells (Ts1). However, the same population of hapten-coupled cells can induce both delayed-type hypersensitivity responses and third-order effector suppressor T cells (Ts3). Thus, splenic and peritoneal antigen-presenting cells from B6.lpr mice display a defined defect in the ability to induce certain suppressor T cell responses. The cellular defect in Ts1 induction is controlled by the lpr gene, since age-matched congenic B6 mice do not display this defect. The splenic adherent cell defect is temporarily correlated with the autoimmunity that develops in B6.lpr animals. The antigen-presenting defect in the B6.lpr splenic adherent population for Ts1 induction is reversible by culturing the cells in interferon-gamma. The results are discussed as an illustration of the relationship between experimental models of autoimmunity and defects in a suppressor T cell cascade.     

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.     

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.     

Cellular origin of blocking factors from cultured spleen cells of tumor-bearing mice

Spleen cells from mice bearing methylcholanthrene-induced tumors were cultured for 2 days without further stimulation. Blocking factors were consistently detected in culture supernatants by their ability to suppress leukocyte adherence inhibition reactions between soluble tumor antigens and peritoneal cells of tumor-bearing mice. The blocking factors were specific for individual tumors. The cellular origin of these factors was investigated by depleting the spleen cell population of various cell types before culturing. The cells involved were removed by treatment with antibodies to certain membrane markers (Thy-1, Ly-2, Ia, I-J) but not by anti-Ly-1 antibodies. Removal of adherent cells also prevented production of blocking factors, which was restored by reconstitution with syngeneic but not allogeneic cells from normal mice. The normal reconstituting cells were shown to bear Ia, but not I-J or IgM. This indicates that blocking factors (previously shown to have I-J determinants in their molecules) originate from suppressor T lymphocytes (Thy-1+, Ly-1-2+, I-J+), with macrophages (I-J-, Ia+) in the role of accessory cells.     

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.     

Cell Surface Expression of I-A Products Is Required for Contact Sensitivity Induction by Trinitrophenyl-Coupled Epidermal Cells

Trinitrophenyl (TNP)-coupled epidermal cells (EC) injected subcutaneously (s.c.) were more capable of inducing contact sensitivity (CS) to 2, 4, 6-trinitro-1-chlorobenzene (TNCB) than similarly substituted spleen cells (TNP-SC). Furthermore, the intravenous (i.v.) or intraperitoneal (i.p.) injection of TNP-EC also induced CS responses, whereas the i.v. or i.p. injection of TNP-SC failed to induce them. Treatment of mice with cyclophosphamide (Cy; 50 mg/kg) or anti I-J serum allowed animals injected with TNP-SC i.v. to develop significant CS responses, suggesting that Cy-sensitive and I-J positive regulatory cells were involved in the induction of unresponsiveness by the i.v. injection of TNP-SC. Mapping studies of the major histocompatibility gene complex (MHC) region demonstrated that identity at the I-A subregion alone between EC donor and recipient mice was sufficient for the induction of CS by TNP-EC given i.v. Blocking experiments using antisera in the absence of complement indicated that I-A subregion-encoded antigens on the surface of TNP-EC apparently are involved in the induction of CS, and are not simply phenotypic markers on the surface of accessory cells.     

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

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

Delayed-type hypersensitivity to hen egg-white lysozyme. I. The surface phenotypes of suppressor T cells induced by intravenous injection of lysozyme-modified spleen cells, and their molecular interactions

Suppressor T cells (Ts) induced by lysozyme-modified syngeneic lymphocytes were characterized. Hen egg-white lysozyme (HEL)-specific delayed-type hypersensitivity (DTH) was suppressed when HEL-induced Ts were transferred into naive mice. These HEL-induced Ts had surface markers of both Thy-1 antigen, and I-J gene products. The suppression of HEL-specific DTH was greatly increased, when these Ts had been enriched with HEL-coated petri dishes. Isolated anti-HEL antibodies from B10.BR or A/Sn mice were inoculated into rabbits to induce anti-cross-reactive idiotype (CRI) antibodies. The rabbit antisera were extensively absorbed with normal B10.BR or A/Sn immunoglobulins (Igs) and MOPC 104E ascites Igs to render them idiotype (Id) specific. Using these anti-CRI antibodies, we observed that these Ts possessed Id receptors on their cell surface. Results of both fluorescence techniques and cytotoxicity tests revealed that about 10% of the enriched T cells containing these Ts were Id positive. Moreover, these enriched T cells were substantially killed by anti-I-J antiserum plus complement. However, this killing was completely blocked by HEL antigen. These results suggest that both Id receptors and I-J gene products might be forming the same molecular complexes or might coexist in the vicinity of the molecule.     

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

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

Characterization of the accessory cells involved in suppressor T cell induction

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.     

Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

Characterization of a cloned ultraviolet radiation (UV)-induced suppressor T cell line that is capable of inhibiting anti-UV tumor-immune responses

Ultraviolet radiation (UV) is a potent carcinogen for the induction of skin tumors. In this regard, UV represents a unique carcinogenic agent, in that depending on the dosage and conditions of administration it can function as either a complete carcinogen, a carcinogenic promoting agent, or an immunologic modulator of anti-tumor rejection responses. The immunologic modulatory activity of UV has been demonstrated in numerous studies. These studies have shown that subcarcinogenic doses of UV induce a population of suppressor T lymphocytes (Ts cells) that allow for the emergence and progression of UV-induced tumors. Although the phenotypic and functional properties of these cells have been established, it was unclear as to whether the UV-induced Ts cell population consisted of multiple Ts cell clones able to recognize a range of unique tumor antigens or a limited number of Ts cell clones with functional specificity directed toward a common tumor-associated antigen (TAA). To address this question, an interleukin 2-dependent, UV-induced cloned Ts cell line was derived, by limiting dilution without exogenous antigen stimulation, from the splenic T cell population of a C3H mouse that had been exposed to a subcarcinogenic dose of UV. This Ts cell line, designated UV2.10, was selected for its ability to suppress the in vitro differentiation of cytotoxic T cells from the draining lymph nodes of UV-induced tumor-immune mice. When transferred into non-UV-irradiated syngeneic mice, which normally reject a UV-induced tumor implant, the UV2.10 cells rendered their hosts susceptible to the growth of a battery of UV-induced tumors. Although capable of suppressing in vitro and in vivo UV-induced tumor-immune responses, UV2.10 cells did not inhibit the elicitation of contact hypersensitivity responses, the rejection of allogeneic skin grafts, responses, the rejection of allogeneic skin grafts, or the rejection of allogeneic UV-induced tumors. These data suggest that the cloned UV2.10 Ts cell line possesses functional antigenic specificity that may be limited to the regulation of immune responses that are directed toward the TAA expressed by syngeneic UV-induced tumors. Employing monoclonal antibodies and FACS analysis, the cell surface phenotype of the UV2.10 cell line was determined to be: Thy-1.2+, Lyt-1-, Lyt-2+/- (dim), L3T4a-, I-A/E-, and I-J+. This cell surface phenotype is indicative of a suppressor T cell. These data lend further support to the hypothesis that the UV-induced Ts cell population is clonal in nature and functions through its ability to recognize a common TAA(s) that appears to be expressed by virtually all UV-induced tumors.     

Characterization and activity of contrasuppressor T cells induced by type III pneumococcal polysaccharide

Optimally immunogenic amounts of type III pneumococcal polysaccharide (S3) activate a population of contrasuppressor T cells (Tcs), which have been shown to play an important role in the induction of anti-S3 antibody responses. These Tcs belong to a unique T cell subset that has the surface phenotype Lyt 1+2- L3T4- I-J+ I-A+. These Tcs are also cyclophosphamide (Cy)-sensitive and sensitive to antilymphocyte serum (ALS) and mitomycin C. Tcs have antigen-binding receptors, indicating that any interactions of Tcs with B cells or T suppressor cells (Ts) (both of which also have antigen-binding receptors) must be via an antigen bridge rather than an idiotype-anti-idiotype interaction. Tcs are also Igh restricted in their action. Contrasuppression is manifest only when the Tcs are Igh compatible with both the Ts and the responding B cells. Tcs apparently mediate their effects by releasing a soluble factor, since a soluble factor extracted from Tcs is able to abrogate the effects of S3-specific Ts.     

Trypanosoma cruzi-induced suppression of IL-2 production. II. Evidence for a role for suppressor cells

Suppression of IL-2 production during experimental Chagas' disease accounts at least in part for the overall depressed state of the immune system in infected mice. The failure to produce IL-2 in response to mitogen stimulation is not the result of the lack of cells capable of producing IL-2, but appears to be due to regulation of IL-2 production by suppressor cells. This conclusion is supported by cell-mixing experiments where the ability of cells from infected mice to suppress normal spleen cell IL-2 production is evident. Although depletion of plastic and Sephadex G-10 adherent cells results in modest increases in IL-2 production by spleen cells from infected mice, even in the presence of normal adherent cells as a source of IL-1 producers, IL-2 production does not approach normal levels. Also, isolated macrophages are not by themselves suppressive for normal spleen cell IL-2 production, whereas plastic and G-10 nonadherent cells from infected mice are. Depletion of Thy-1+ and Ly-2+ cells not only completely abrogates the ability of spleen cells from infected mice to suppress normal IL-2 production, but results in a cell preparation which actually enhances IL-2 production. Anti-Ly-2 and C treatment of infected spleen cells also markedly enhances their ability to produce IL-2. These results indicate a major role for Ts cells in the regulation of IL-2 production, and a relatively minor role of macrophages as direct effector cells of suppression in this response. The ability to enhance IL-2 production in this system with PG synthesis inhibitors suggests a role for PG-producing cells such as macrophages in the suppressor mechanism, perhaps as inducers of the suppressor effector cells.     

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

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