Wlodzimierz Ptak

Epicutaneous immunization induces αβ T-cell receptor CD4 CD8 double-positive non-specific suppressor T cells that inhibit contact sensitivity via transforming growth factor-β

Since it was previously shown that protein antigens applied epicutaneously in mice induce allergic dermatitis mediated by production of T helper 2 (Th2) cytokines we postulated that this might induce suppression of Th1 immunity. Here we show that epicutaneous immunization of normal mice with a different protein antigen applied on the skin in the form of a patch induces a state of subsequent antigen‐non‐specific unresponsiveness caused by suppressor T cells (Ts) that inhibit sensitization and elicitation of effector T‐cell responses. Suppression is transferable in vivo by αβ‐T‐cell receptor CD4+u2003CD8+ double positive lymphocytes harvested from lymphoid organs of skin patched animals and are not major histocompatibility complex‐restricted nor antigen specific. Both CD25+ and CD25–u2003CD4+u2003CD8+ T cells are able to suppress adoptive transfer of Th1 effector cells mediating cutaneous contact sensitivity. In vivo treatment with monoclonal antibodies showed that the cytokines interleukin (IL)‐4, IL‐10 and transforming growth factor‐β (TGF‐β) are involved in the induction of the Ts cells. Additionally, using IL‐10–/– mice we found that IL‐10 is involved in skin induced tolerance. Further in vitro experiments showed that lymph node cells of skin tolerized mice non‐specifically suppress [3H]thymidine incorporation by antigen‐stimulated immune cells and this effect can be abolished by adding anti‐TGF‐β, but not anti‐IL‐4 nor anti‐IL‐10 antibodies. These studies indicate the crucial role of TGF‐β in skin induced tolerance due to non‐antigen‐specific Ts cells and also show that IL‐4, IL‐10 and TGF‐β play an important role in the induction of epicutaneously induced Ts cell suppression.

Epicutaneous Application of Protein Antigens Incorporated into Cosmetic Cream Induces Antigen-Nonspecific Unresponsiveness in Mice and Affects the Cell-Mediated Immune Response

Background: Protein antigens applied epicutaneously by the patch method induce allergic dermatitis mediated by IgE antibodies in mice and simultaneously significant suppression of Th1-mediated delayed-type hypersensitivity (DTH) reactions. Methods: We developed a method in which protein antigens (calf collagen, elastin, keratin, TNP-substituted mouse Ig) were homogenized with neutral cream. Animals treated epicutaneously with such preparations were tested for contact sensitivity to TNP hapten or DTH hypersensitivity to hemocyanin. Antigen specificity of induced unresponsiveness was tested in vivo in ‘transfer-in’ and ‘transfer-out’ experiments. The influence of skin-induced regulatory cells on in vitro [3H]thymidine uptake by immune cells as well as the possible mode of their action using anticytokine antibodies were tested. Results: Our procedure in which different protein antigens are applied on the skin in the form of cream induces a state of antigen-nonspecific unresponsiveness affecting cell-mediated immune responses in mice. Cream alone has no such effect. Suppression is transferable in vivo by TCR-αβ lymphocytes, while Tγδ cells show no activity. In vitro, lymphoid cells of skin-tolerized mice suppress [3H]-thymidine incorporation by immune cells and this effect can be abolished by adding anti-TGF-β but neither anti-IL-4 nor anti-IL-10 antibodies. Conclusions: Lack of antigen-specifity of unresponsiveness induced by epicutaneous deposition of cream containing protein antigens resembles ‘determinant spreading’ in oral tolerance induced by antigen feeding. This may suggest that similar immunoregulatory mechanisms operate on these two bodily surfaces.

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.

Serotonin Initiation of Delayed-Type Hypersensitivity: Mediation by a Primitive Thy-1+ Antigen-Specific Clone or by Specific Monoclonal IgE Antibody

Elicitation of delayed-type hypersensitivity (DTH) responses is due to the required sequential action of two different antigen (Ag)-specific Thy-1+ cells: early acting, DTH-initiating cells and locally recruited CD4+, alpha beta-TCR+, DTH effector T cells. DTH-initiating cells have an unusual phenotype for Ag-specific cells (Thy-1+, CD5+, CD4-, CD8-, CD3-, sIg-, B220+ (CD45RA+), Mac 1+, IL-2R- and IL-3R+) and act by producing Ag-specific non-IgE factors that sensitize mast cells for release of the vasoactive amine serotonin (5HT) at the local site of elicitation of DTH by challenge. Another mechanism of DTH initiation involves Ag-specific IgE antibodies that also can sensitize mast cells for local serotonin release. Serotonin initiates DTH by activating the local endothelial cells to allow recruitment of DTH effector T cells, and also by activating 5HT-2 receptors on these recruited T cells.

Epicutaneous Immunization with Protein Antigen in the Presence of TLR4 Ligand Induces TCRαβ+CD4+ T Contrasuppressor Cells That Reverse Skin-Induced Suppression of Th1-Mediated Contact Sensitivity

Our previous work showed that epicutaneous (EC) immunization of mice with different protein Ags applied on the skin in the form of a patch induces a state of subsequent Ag-nonspecific unresponsiveness due to suppressor CD4+8+ T cells (Ts) that inhibit Th1-mediated contact sensitivity (CS) reactions via released TGF-β. In the present work we show that EC immunization with Ag together with the TLR4 ligand LPS induced cells that could prevent suppression by the Ag-nonspecific Ts. These up-regulatory cells, called contrasuppressor T cells (Tcs), belong to a population of Ag-specific TCRαβ CD4+ lymphocytes and are different from Th1 CD4+ cells that mediate the CS reaction. Experiments using knockout mice showed that EC induced contrasuppression is MyD88, INF-γ, and IL-12 dependent, whereas IL-6 is not involved in this phenomenon. Additional experiments with anti-IFN-γ mAb showed that IFN-γ is required for induction of Tcs cells but does not play a crucial role in the effector phase of contrasuppression. Additionally, treatment of CS effector cells with rIL-12 makes them resistant to EC induced suppression without affecting Ts cells, whereas IL-12 neutralization in vitro abrogates contrasuppression. These data show that IL-12 is indeed involved in the effector phase of EC induced contrasuppression and that this cytokine does not act directly on Ts cells. The mechanism of action of Tcs protects Th1 effector cells mediating CS from the nonspecific Ts, leaving suppression to other Ags intact. Ts and Tcs cells do not influence each other and can be induced simultaneously in the same animal.

Positive Regulatory γδ T Cells in Contact Sensitivity: Augmented Responses by in Vivo Treatment with Anti-γδ Monoclonal Antibody, or Anti-Vγ5 or Vδ4

Contact sensitivity (CS) responses, induced by skin painting with reactive haptens like picryl chloride or oxazolone, are classical examples of in vivo immunity mediated by αβ T cells. Our previous studies showed that γδ T cells were required to assist the αβ CS-effector T cells in the successful adoptive cell transfer of CS responses. These spleen and lymph node-derived γδ+ CS-assisting regulatory cells were CD3+, CD4-CD8+, non-antigen-specific, and non-MHC-restricted, and preferentially expressed Vγ5 and Vδ4 variable regions.In the current study we show that systemic treatment of mice in vivo with anti-γδ mAb, produced a similar positive influence on CS responses in two different systems: i.e. active sensitization, or adoptive cell transfer. In addition to augmented CS responses produced by treatment with pan anti-γδ TCR mAb, anti-γδ-V region mAb were examined, and augmentation of CS also was produced by anti-Vγ5 and anti-Vδ4 mAb, the V regions determined previously to be preferentially expressed on γδ ...

Preferential Induction of Antigen-Specific Contrasuppressor T Lymphocytes by Trinitrophenyl (TNP)-Substituted Langerhans Cells

We have tested the ability of trinitrophenyl (TNP)‐labelled antigen‐presenting cells (Langerhans cells (LC) and peritoneal macrophages (Mø)) administered intravenously to induce cells that mediate and regulate contact sensitivity. TNP‐Mφ fail to induce contrasuppressor cells (Tcs) but activate efferent T suppressor (Ts) cells. However, the activity of immune cells can be recovered after removal of Ly 2 Ts cells. In sharp contrast, TNP‐substituted purified LC produced a significant contact sensitivity reaction, which is roughly equivalent to that achieved by skin sensitization with picryl chloride. Immune cells from these animals were relatively resistant to suppression by antigen‐specific Ts cells, and we have found that their resistance is due to the presence of Ly 1, Vicia villosa lectin adherent, I‐J+ cells. The Tcs cell induced by TNP‐LC is indistinguishable by surface markers and function from Tcs cell found in mice injected with antigen‐antibody complexes. Both these Tcs cells are capable of protecting immune cells from the effects of suppression by antigen‐specific Ts cells if added in the proper sequence. Although they have identical surface phenotypes, it is not known at present whether or not Tcs cells induced by two different antigen presentations are identical. The possible reasons why LC are such potent inducers of contrasuppression are discussed.

Cell Interactions that Regulate the T-Cell Dose-Response Profile to Concanavalin A

We have tested the ability of structures on macrophage (Mφ) membranes (Mφ‐MEM) and on several other types of cells with Fc receptors to affect the DNA synthetic response of concanavalin‐A‐stimulated T cells. Of the cells tested, only Mφ‐MEM have the capacity to relieve the suppression produced by supra‐optimal doses of the mitogen. The Mφ‐MEM do not increase Con A responses by altering the stimulated capacity of the Con A. These observations, analysed together with previous results, which have indicated that live intact Mφ are required for the transfer of information between lymphocyte sets and that Mφ‐MEM preparations can act as competitive antagonists for this function, are interpreted as follows: some supraoptimal doses of Con A activate suppressor cells, which are responsible for limiting the DNA synthetic response to the mitogen; the Mφ‐MEM abrogate this suppression by absorbing the signal that activates the suppressor cell. Kinetic studies suggest that the Mφ‐MEM do not affect the activity of already activated suppressor cells. We also found that Con A usually activates two separately responding T‐cell populations with different sensitivities to dose and to time of contact with mitogen and that the suppression of both populations can be relieved by Mφ‐MEM. These results support the notion that the overall immunological circuit is composed of multiple independently regulated mini‐circuits, with Mφ acting as transmission posts for intra‐ and perhaps also inter‐circuit communication.

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.

Suppression and contrasuppression in athymic nude mice: Nude mice produce the antigen-specific component of a T suppressor factor that inhibits the late 24-hr phase of DTH, but do not generate suppression nor contrasuppression of the early initiating phase of DTH☆

Previous studies demonstrated that the initiation of murine delayed-type hypersensitivity (DTH), as exemplified by contact sensitivity induced by picryl chloride (PCI) or oxazolone (OX), is due to antigen-specific, T cell-derived, DTH-initiating factors called, respectively, PCl-F and OX-F. These factors participate in the extravascular recruitment of CD4+, Th-1, DTH effector T cells in the elicitation of DTH. Related factors also participate, together with nonantigen binding factors derived from CD8+ T cells, to constitute an antigen-specific T cell-derived suppressor factor (TsF) that can down regulate the ability of Th-1 effector T cells to mediate DTH. Since it was shown recently that athymic nude mice can make antigen-specific, DTH-initiating T cell factors, the current study tested whether nude mice also could produce the antigen-specific component of the TsF that suppresses DTH effector T cells. We found that antigen-specific factors from nu/nu mice could complement the nonantigen-binding subfactor produced in normal mice to constitute the whole antigen-specific TsF. Additional studies showed that the successful adoptive cell transfer of DTH-initiating T cell activity from nude mice into normal mice required cyclophosphamide treatment of the recipient. In contrast, transfer of DTH-initiating cell activity from nu/+ mice did not require cyclophosphamide treatment of the recipients. We hypothesized that nude mice lacked contrasuppressor cells. Although nude mice were able to manifest the early, initiating phase of DTH, we found that there was no suppression of early DTH-initiating T cells in nude mice, compared to nu/+. Therefore the production of DTH-initiating T cell factor could be boosted in nude mice. The ability to boost DTH-initiating cells in nude mice should facilitate the development of cell lines and clones with the ability to initiate DTH.