Intermediary role of macrophages in the passage of suppressor signals between T-cell subsets.

We have examined the ability of macrophages (Mphi) to transmit T-cell derived suppressor signals to other T cells. The suppressor signal studied is an antigen-specific factor which suppresses the ability of adoptively transferred, sensitized lymphocytes to express contact hypersensitivity in normal recipients. We have found that this factor binds to peritoneal exudate Mphi via cell surface structures which can be blocked with heat-aggregated gamma globulin. Dead (HK) Mphi bind the factor but fail to present it in a functional way to assay (immune) T cells, whereas live (L) Mphi perform both functions. Further, L Mphi can retrieve the factor in an active form from the surfaces of HK Mphi. Based on these and other findings (1-5), we discuss the possibility that Mphi may play as important a role in presenting T-cell communication signals to the cells of the immune system as they do in presenting antigen.

Mechanisms of suppression in the transfer of contact sensitivity. Analysis of an I-J+ molecule required for Ly2 suppressor cell activity.

The passive transfer of contact sensitivity (CS) by immune cells can be inhibited with an antigen-specific T suppressor factor. This factor is composed of two subfactors: an antigen-specific subfactor made by an Ly1+ cell (PC1-F) and a antigen nonspecific subfactor made by an Ly2+ T cell (TNBSA-F). The suppressive activity of the complete factor can be eliminated by depleting the assay population of Ly2+ cells, even though it is the Ly1+ cell in the population that transfers the adoptive immunity. This suggests that the Ly2+ cell in the assay population is needed to transduce the suppressive signal to the Ly1+ effector cell of DTH. We found that an Ly2+ cell from immune animals could be induced to produce a cell free subfactor that overcame the requirement for this Ttrans cell in the suppression of CS by TsF. The induction required only PC1-F, TNP-coupled spleen cells, and resulted in the production of an antigen-nonspecific I-J+ subfactor by immune Ly2+, I-J+ cells. The need for the Ly2+ transducer cell could also be overcome by addition of an I-J+ molecule secreted by Ly1 T cells hyperimmunized to SRBC. A suppressor complex made from mixing the I-J+ molecule with TNBSA-F could directly suppress the functional activity of immune T cells not only to transfer CS, but also to deliver help to B cells in an in vitro PFC response. This suppressive complex is antigen-nonspecific and does not require Ly2+ T cells in the assay population for suppressive activity. These results indicate that effector factors of the suppressor circuit require two molecules; one that contains the functional suppressor material and one that serves as a "schlepper," a molecule needed to deliver the suppression to the appropriate target cell. The ability to construct a functional suppressor complex from two subfactors raised against different antigens, using different immunization procedures, which were isolated from factors exhibiting different functional activities suggests that certain cells of the immune system may play a universal role in "transducing" the suppressive signal.

T cells produce an antigen-binding factor with in vivo activity analogous to IgE antibody.

T cell-dependent activation of resident tissue mast cells is required for the elicitation of delayed-type hypersensitivity skin reactions in mice. A T cell-derived antigen-binding factor that transfers the ability to elicit an immediate hypersensitivity-like skin reaction is described and compared with a hybridoma IgE antibody. Both the T cell factor and IgE mediate reactions with increased vascular permeability and both are mast cell dependent, as they are inactive in two different types of mast cell deficient mice (W/Wv and Sl/Sld). The T cell factor was distinguished from IgE by affinity chromatography using specific anti-IgE and anti-factor antibodies and by a shorter duration of passive sensitization. The T cell factor is a suitable candidate for participation in the mechanism by which T cells activate mast cells in delayed-type hypersensitivity.

Role of antigen-presenting cells in the development and persistence of contact hypersensitivity.

Three outcomes pertinent to contact sensitivity (CS) follow immunization with various forms of trinitrophenylated (TNP) substrates: (a) specific immunological unresponsiveness for CS is induced when immunization favors activation of splenic suppressor cells. This state is achieved by intravenous injection of trinitrophenyl-conjugated to various types of cells, such as peritoneal exudate cells (PEC). (b) A short-lived or evanescent form of CS is induced when immunization reduces activation of the suppressor circuit. This can be achieved by subcutaneous immunization with trinitrophenyl conjugated to syngeneic PEC, by pretreatment with cyclophosphamide to diminish suppression before intravenous immunization, or by altering the mode of antigen presentation by using TNP-substrate that has undergone phagocytosis. (c) A long-lived form of CS is induced when trinitrophenyl is presented to the immune system on skin cells either by contact skin painting with reactive trinitrophenyl, or by subcutaneous, or even intravenous injection of trinitrophenyl-conjugated epidermal cells. In fact, trinitrophenyl-conjugated epidermal cells induced CS even when the suppressor circuit was activated by intravenous coadministration of TNP-PEC. This implies that antigen presentation on epidermal cells induces sensitized cells that are relatively resistant to suppression. The cell type(s) in the skin that are primarily responsible for this potent form of antigen presentation are most likely Langerhans cells, because they can be concentrated by virtue of their Fc receptors and they are Ia positive. Thus, both the anatomical site where antigen is first encountered by the immune apparatus, as well as the nature of the cells which present the antigen, determine whether a CS response will ensue, as well as whether it will be evanescent or long-lasting.

Role of contrasuppression in the adoptive transfer of immunity.

The data presented in this paper show that the population of cells that adoptively transfer contact hypersensitivity are Lyt-1+ 2-, I-J- and nonadherent to V. villosa lectin. However, the adoptive transfer of immunity by this population of cells is successful only when the recipient has been treated in such a way as to impair the host immunosuppression mechanism. This population cannot, on its own, transfer immunity to adult, untreated naive recipients unless an additional population of immunoregulatory cells is present. This immunoregulatory population does not itself adoptively transfer immunity. This latter population is differentiated from the immune cells in that they are Lyt-1+ 2-, I-J+ and are adherent to V. villosa lectin. Both populations are required to adoptively transfer immunity to adult untreated naive recipients.

Specialized antigen-presenting cells. Splenic dendritic cells and peritoneal-exudate cells induced by mycobacteria activate effector T cells that are resistant to suppression.

We have tested the ability of several types of trinitrophenyl (TNP)-labeled Ia+ cells to induce contact hypersensitivity (CS) after intravenous injection. Most labeled cell types (spleen cells, splenic macrophages, various types of peritoneal-exudate cells) not only fail to induce CS after this type of inoculation but, rather, activate T suppressor cells leading to specific immunological tolerance. Occasionally, some of these immunizing cells managed to bypass the T suppressor system and induced CS. In those cases the response was short-lived and could be blocked by concomitant injection of trinitrobenzelsulphonic acid (TNBS), a potent inducer of T suppressor cells. In sharp contrast to these results, TNP-labeled splenic dendritic cells and TNP-labeled peritoneal-exudate cells induced by complete Freund's adjuvant had the following distinctive features: (a) They were always able to sensitize when injected intravenously, and the degree of sensitization they produced was roughly equivalent to that achieved by cutaneous application of picryl chloride, the chemically reactive form of TNP. (b) The response they elicited was long lived (i.e., lasted for greater than 3 wk). (c) Their sensitizing capacity could not be blocked by the concomitant injection of TNBS. (d) They elicited a response that could be adoptively transferred to untreated, normal recipients. These results indicate that the type of cell that first presents antigen to the immune system plays an important, even essential, role in determining the strength and duration of the subsequent immune response. In particular, the results suggest that some special antigen-presenting cells can induce a response that is relatively resistant to host suppressor mechanisms. Evidence that they do so by activating contrasuppressor cells is discussed.

Gamma delta T cells from tolerized alpha beta T cell receptor (TCR)-deficient mice inhibit contact sensitivity-effector T cells in vivo, and their interferon-gamma production in vitro.

Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using alpha beta T cell-deficient (TCR alpha -/-) mice, are presented here. The results clearly show that TCR alpha -/- mice cannot mount CS, implicating alpha beta T cells as the critical CS-effector cells. However, TCR alpha -/- mice can, after high dose tolerance, downregulate alpha +/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized alpha -/- mice are shown to include gamma delta TCR+ cells that also can downregulate interferon-gamma production by the targeted CS-effector cells in vitro. Downregulation by gamma delta cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that gamma delta T cells cannot fulfill CS-effector functions performed by alpha beta T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by gamma delta T cells. Comparisons are likewise considered with downregulation by gamma delta T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.

The influence of collagenase treatment on the production of TNF-alpha, IL-6 and IL-10 by testicular macrophages.

Testicular macrophages (TMf) are located in the interstitial tissue of the male gonad. Highly purified TMf populations can be prepared either by the mechanical shaking of dispersed testicular tissues or by enzymatic digestion with collagenase followed by cell adherence, rosetting and gradient centrifugation. TMf obtained by the enzymatic procedure produced significantly more cytokines (IL-6, IL-10 and TNF-alpha) than TMf isolated by the mechanical method and this effect is long-lasting. Our results indicate that isolation of tissue macrophages by enzymatic digestion may influence their functional activity, and suggest that critical evaluation of the method used to obtain these cells should be the regular practice.

Distinct populations of antigen-presenting macrophages are required for induction of effector and regulatory cells in contact sensitivity response in mice.

Macrophages (Mf) and other antigen-presenting cells (APCs) are able to induce both immune and regulatory T cells. We compared the antigen-presenting activities of different subpopulations of thioglycolate-induced peritoneal macrophages from mice that were or were not treated with cyclophosphamide (CY) by several functional (adherence and phagocytosis) and morphologic (phenotypic) markers (FcR, Ia). Different subpopulations of macrophages were derivatized with trinitrophenyl and injected intravenously into recipients, which were tested directly for a contact sensitivity (CS) reaction or for the presence of efferent suppressor T (Ts) cells in passive transfer experiments. Our results demonstrate that peritoneal macrophages are both morphologically and functionally heterogeneous. Macrophages that induce immune cells that mediate CS have characteristics different from those that induce Ts cells. They accumulate in the low-density cell fraction on a discontinuous Ficoll gradient, are insensitive to treatment in vivo with low doses of CY, phagocytose poorly and adhere to plastic, and perhaps have low expression of FcRI and FcRII. Both macrophage fractions seem not to differ in expression of Ia, Mac-1, and Mac-3 antigens. It is argued that low doses of CY abrogate suppression in vivo by selective action on Ts cells. Our results confirm that at least a portion of the action of CY may be due to its influence on certain subpopulations of APCs.

Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice.

The choice of the type of instrument to measure delayed-type hypersensitivity (DTH) in mice, as assayed by ear swelling reactions, influences the experimental results. When a caliper that applies little pressure to the ears is employed, DTH reactions in ears of mice sensitized to picryl chloride show an early onset at 2 h after challenge, comparable swelling at 4 h and a slow rise to a 24 h classical peak response thereafter. In contrast, 3 different micrometers that apply more pressure to the ears reveal a biphasic pattern of ear swelling reactions in mice immunized and challenged with picryl chloride. The early component of DTH measured by these micrometers peaks 2 h after challenge. Thereafter the measured ear thickness declines, and the onset of the classical delayed reaction is detected at 12 h after ear challenge. Yet another instrument, that in contrast to the caliper and micrometers mentioned above, applies all the pressure to only a very restricted area of the ear, fails to detect an early swelling reaction; the delayed reaction is first detected at 12 h after ear challenge and rises thereafter to a 24 h peak. The differences in outcome of the assays using the different instruments indicate that the early component or DTH reactions differs from the late component of DTH reactions in that the early swelling is easier to compress when pressure is applied by the instrument used for measurement. This is probably caused by the fact that the late reactions are due to a cellular infiltrate, whereas the early reactions are edematous in character, and are due to accumulation of plasma components.

Fetal suppressor cells. Their influence on the cell-mediated immune responses.

The cell-mediated immune responses are significantly reduced in fetal and newborn mice, furthermore, spleen cells of these animals are able to suppress the immune responses of adult lymphocytes in a variety of situations. Newborn mice sensitized to picryl chloride within 24 hr after birth fail to develop contact sensitivity reaction when tested several weeks later, and fetal mice do not develop graft-versus-host reaction when given injections of parental lymphocytes. Spleen cells of fetal or newborn mice are able to suppress the passive transfer of contact sensitivity and the local graft-versus-host reaction elicited by immunized parental cells in F1 hybrid mice, and reduce significantly the severity of graft-versus-host reaction and mortality rate in cyclophosphamide-treated F1 recipients. In no experiment were thymus cells of either fetal or newborn mice found to be inhibitory. The possible mechanisms of action and biological significance of fetal suppressor cells are discussed.

Cytotoxic macrophage in graft-versus-host reaction.

Peritoneal macrophages of F1 hybrid mice injected with parental strain spleen cells 8-14 days previously ("early" graft-versus-host (GVH) macrophages) develop an increased ability to destroy in vitro bystanding target cells of syngeneic, allogeneic, and xenogeneic origin. At later stages of GVH reaction, the nonspecific cytotoxicity of macrophages wanes ("late" GVH macrophages) and does not differ much from that of control macrophages. Trypsinization of early GVH macrophages or iodoacetate treatment significantly reduces their cytotoxicity, whereas anti-theta serum and complement have only a moderate effect. Antimouse IgM antibody significantly enhanced the cytotoxic potential of early GVH macrophages, but had no effect either on normal or late GVH macrophages. In contrast, antimouse IgG antibody enhanced, although slightly, only the cytotoxic activity of late GVH macrophages. Two possible explanations of the increased cytotoxicity of early GVH macrophages are offered: (1) cytophilic antibody of IgM type, directed against host antigens, renders macrophages capable of damaging nonspecifically bystanding target cells upon contact with antigen; (2) IgM alloantibody produced by parental cells changes the surface properties of macrophages and contributes to the cytotoxicity of these cells.

Contrasuppression and tumor rejection.

The growth of a highly progressive MCA-induced tumor 3152-PRO is dependent on the activity of suppressor T cells (Ts). Injection of syngeneic mice with antibodies specific for Ts leads to enhanced tumor transplantation resistance of the 3152-PRO tumor. In addition, injection of recipient mice with highly immunogenic regressor tumors conjugated with trinitrophenyl (TNP) activates a T cell population which also mediates protection to transplantation of TNP-conjugated 3152-PRO tumor cells. One such tumor, 1591-RE, was investigated in detail to determine the phenotype and biologic activity of this T cell population in overcoming Ts cell activity. Induction of transplantation resistance requires the presence of TNP hapten on both the highly regressive immunizing tumor (and not its progressor variant 1591-PRO4), and on the challenge tumor 3152-PRO. The cell population from TNP-1591-RE immunized animals which mediates protection against the transplantation of TNP-3152-PRO is Thy-1+, CD4+, 8-, Lyt1+, I-J+, and Vicia villosa lectin adherent, the identical phenotype to antigen-specific contrasuppressor T cells in the contact sensitivity (CS) response to TNP in vivo. A T cell population of identical phenotype from TNP-1591-RE immunized mice can overcome the effects of antigen-specific Ts cells on PCl-immune cells in the adoptive transfer of CS in vivo. These results suggest that immunoregulatory cells that mediate protection against progressive tumors may be identical in function to antigen-specific contrasuppressor T cells.

Induction of "allogeneic effect"-like reaction by syngeneic TNP-modified lymphoid cells.

TNP-substituted SRBC-immune spleen cells, when injected into cyclophosphamide-treated recipients, are recognized by T lymphocytes and produce, in the presence of specific antigen (SRBC), significantly more PFC than nonsubstituted cells. Labeling of immune B cells is more important in producing the augmented responses than is the labeling of immune T cells. TNP determinant has to be bound directly to the transferred immune cells to produce enhanced antibody responses, as when recipients were injected with non-substituted immune cells and TNP-substituted non-immune cells simultaneously, no increase in PFC number was noted (lack of a bystander effect). When recipients were rendered tolerant to TNP, two separate effects were observed, dependent on the mode of inducing unresponsiveness. In mice which were treated with TNP over an extended period of time, lack of recognition of TNP was demonstrated, such that TNP-substituted cells failed, when transferred, to produce an augmented response. When a short-term tolerogenic regime was used, the adoptively transferred TNP-labeled cells gave a very poor response (greater than 95% inhibition) due to in vivo suppression and/or killing. These results, together with the lack of influence of tolerance induced to unrelated hapten (DNP or OX), confirm the antigen specificity of the phenomenon. The reaction observed by us shows a striking resemblance with, but not identical to, the "allogeneic effect" produced by MHC encoded alloantigens. Our results extend the list of analogous immune reactions induced by MHC encoded alloantigens and TNP-derivatized self.

Induction of suppressor cells and cells producing antigen-specific suppressor factors by haptens bound to self carriers.

Injection of TNP-, DNP- or oxazolone-substituted syngeneic cells into mice causes the development of hapten-specific T suppressor cells which prevent the animals from being activity sensitized with homologous hapten. These cells injected together with immunized cells abrogate the latter's ability to transfer passively the contact sensitivity (CS) reaction into normal recipients. T lymphocytes from animals made unresponsive and sensitized with homologous hapten synthesize in vitro antigen-specific suppressor factors (SF) which when incubated with immune lymphocytes prevent them transferring adoptively the CS reaction. The type of cell used to induce suppression or production of suppressor factor (haptenated erythrocytes, thymocytes or macrophages) is not critical suggesting that a hapten-substituted common membrane structure is recognized as a tolerogen. The present work demonstrates that while the specific unresponsiveness induced by cell-bound hapten in vivo is long lasting, cells from tolerized animals are able to suppress the immunized cells in passive transfer or produce in vitro antigen-specific suppressor factors only when tested several days after tolerization.

The effect of antibody on the migratory pattern of peritoneal macrophages.

51Cr-labeled peritoneal macrophages which interiorized sheep erythrocytes (SRBC) injected i.p. into syngeneic recipients accumulate in a typical fashion in different organs. The pattern of distribution can be altered by two maneuvers--when animals at the time of macrophages transfer are injected i.v. with cytophilic antibody, or when macrophages are fed with IgG-SRBC complexes instead of native SRBC. The possible role of modified migratory pattern of macrophages in antibody-induced suppression of the immune response is discussed.

Feedback inhibition of IgM memory cells by high antigen dose.

The ability of high and low antigen doses (SRBC) to recruit IgM memory cells has been compared in several strains of mice. In intact animals priming with low doses was more efficient than priming with high doses. If, however, mice of different strains were X-irradiated two days after priming and repopulated, regardless of whether syngeneic or allogeneic splenocytes were used, they fell into two categories--those in which more memory cells were found after low antigen priming and those in which the reverse was true (Swiss mice). Two possible explanations are offered to explain these interstrain differences--antibody mediated suppression, and generation of suppressor T cells. Our data favor the latter, and we assume that suppressor T cells appear at different intervals after priming in different strains of mice, and that these cells are radioresistant.

Splenic suppressor cells in fetal and newborn mice.

Newborn mice are refractory to sensitization with picryl chloride and the graft versus host reaction (GvHR) in F1 fetal mice injected with parental lymphocytes is markedly reduced. Moreover, spleen cells (but not thymic lymphocytes) of fetal or newborn animals are able to suppress the cell-mediated immune responses, contact sensitivity (CS) and GvHR in a variety of experimental situations. The inhibitory cells are thy-positive and their suppressive activity wanes early after birth.

Primary immune response to sheep red blood cells in mice treated with immunoregulatory alpha-globulins (IRA).

Alpha-Globulins from human and bovine sera or from mouse ascitic fluid were separated by ion-exchange chromatography on DEAE-cellulose into fractions A, B, and C. Fractions A and B had no immunosuppressive activity; fraction C injected into mice at the time of antigen administration, but not later, significantly reduced the number of anti-SRBC plaque-forming cells in the spleens of experimental animals. Single high dose inhibited the response better than the same doses given on 4 consecutive days. It is concluded that a critical level of alpha-globulins is necessary to render lymphocytes hyporesponsive, and when once stimulated by antigen they become less susceptible to alpha-globulin action.

Generation of anti-hapten T cell cytotoxicity in vivo. Relationship to contact sensitivity and the role of contrasuppression.

Immunization procedures that induce contact sensitivity to the trinitrophenyl (TNP) hapten in vivo were investigated for their ability to induce TNP-specific cytotoxic T lymphocytes in vivo. Spleen cells from C3H/HeN mice primed for CS responses either by the topical application of picryl chloride or by the adoptive transfer of PCL immune cells show little or no cytolytic activity in vitro against TNP-coupled target cells. Intravenous immunization with TNP-substituted syngeneic spleen cells, a procedure known to make animals unresponsive to agents normally inducing CS, also failed to induce cytolytic activity in spleen cells. However, both PCL sensitization and adoptive transfer, when combined with the injection of TNP-substituted syngeneic spleen cells, induce significant cytolytic activity against TNP-haptenated BW5147 target cells in vitro. Furthermore, i.v. injection of TNP-spleen cells with surface-bound immune complexes of the IgM or IgG1 isotypes, or with a monoclonal TNP-specific contrasuppressor T cell factor also induces strong antigen-specific cytolytic activity against TNP modified targets. TcsF bears serological determinants of T cell receptor alpha and beta chains and adheres to specific antigen columns. All these immunization regimens were shown to induce CS to TNP as well as the generation of contrasuppressor T cells. The CTL generated in the spleens of immunized mice are Thy1+ CD8+ T cells an are antigen-specific and genetically restricted. The implications of these results with respect to the mechanisms by which cytolytic responses are controlled in vivo is discussed.