Geoffrey L. Asherson

Production of immunity and unresponsiveness in the mouse by feeding contact sensitizing agents and the role of suppressor cells in the Peyer's patches, mesenteric lymph nodes and other lymphoid tissues

Abstract Mice were fed the contact sensitizing agents “oxazolone” or picryl chloride by tube. A single feed gave rise to contact sensitivity. However, the contact sensitivity and antibody production which occurred in mice painted with oxazolone were almost abolished when the mice were fed oxazolone 14 days before the skin painting. Feeding also reduced the DNA synthesis response in the regional lymph nodes. Two types of suppressor cells were found in mice after feeding. After a single feed of picryl chloride the Peyers patches and mesenteric lymph nodes contained suppressor cells which suppressed the passive transfer of contact sensitivity. After three feeds of either agent spleen cells also caused inhibition. These suppressor cells were presumptive B cells as shown by their ability to form rosettes with red cells coated with antibody and complement and their resistance to anti-θ serum and complement. However, separated T cells from the same spleen transferred contact sensitivity. In addition to these B suppressor cells the spleens and peripheral lymph node cells of mice fed with contact sensitizing agent and then painted on the skin contained T cells which limited DNA synthesis in lymph nodes. This was shown by injecting their cells into normal recipients which were then painted with contact sensitizing agent and measuring DNA synthesis 4 days later in the regional lymph nodes. It was concluded that suppressor B and T cells were an important part of the mechanism of unresponsiveness caused by feeding contact sensitizing agents.

Contact sensitivity to picryl chloride: The occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice

Abstract Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus. T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.

T-cell depletion of allogeneic bone marrow prevents acceleration of graft-versus-host disease induced by exogenous interleukin 2

Highly purified human recombinant interleukin 2 (IL-2) markedly accelerated lethal GVHD in the H-2-identical B10.BR----CBA combination, but had no effect when the donor cells were depleted of mature (Thy-1.2-positive) T lymphocytes, indicating a strong immunopotentiating effect of IL-2 on mature T cells causing GVHD. In the same donor-host combination, IL-2 did not influence the recovery from the post-transplantation bone marrow aplasia. The results suggest that IL-2 could be considered for adjuvant hormonal therapy to enhance immune recovery in recipients of T-cell-depleted allogeneic marrow.

Role of IL-4 in delayed type hypersensitivity

IL‐4 plays a key role in the contact sensitivity skin reaction. This has several implications. First, the view that contact sensitivity (CS) is only mediated by cells with a Th1 profile of cytokine secretion needs modification, in the light of the essential role of IL‐4 at the effector stage. Second, the concept of a single cell involved in the systemic transfer of CS is no longer tenable, as it is known that both αβ and γδ cells are required. Studies with the cell lines (which contain both αβ and a few γδ cells) suggest that this double requirement may involve the action of IL‐4 on γδ cells, which bear receptors for IL‐4. Finally, the view that T cell lines only transfer CS when injected locally, but not when injected intravenously (systemic transfer), is correct but incomplete, as T cell lines actually give systemic transfer of CS, providing the cell line or the recipient is treated with IL‐4.

THE ROLE OF THE T ACCEPTOR CELL IN SUPPRESSOR SYSTEMS: Antigen-Specific T Suppressor Factor Acts via a T Acceptor Cell; This Releases a Nonspecific Inhibitor of the Transfer of Contact Sensitivity When Exposed to Antigen in the Context of I-J

The development of our knowledge of suppressor cell systems that influence contact and delayed hypersensitivity may be divided into five stages. The first stage was the recognition of suppressor cells 1. a and the realization that T suppressor cells limited contact and delayed hypersensitivity.1, The second stage was the observation that suppressor cells made antigen-specific The other stages were the recognition of several types of suppressor cells, some of which only acted when given early in the immune response (Ts-aff) and others of which acted at the expression stage in a 24-h experiment (Tseff) ; 6-8 the recognition that some suppressor cells were antigen-driven, while others were anti-idiotype-driven; and the recognition of genetic restrictions in the action of suppressor cells and suppressor factors involving the major histocompatibility complex and, in the case of anti-idiotype suppressor cells, the Igh-V locus that controls the variable region of the immunoglobulin molecules and antigen-specific T cell f a c t o r ~ . l ~ ~ ~ Recently, attention has shifted to the modes of interaction among the various T suppressor cells. In the idiotype-anti-idiotype link, either a T suppressor cell, variously called Ts,, Tsi, or Ts-aff, or an antigen-specific T suppressor factor gives rise to an anti-idiotype suppressor cell by a process of immunization. In the acceptor cell link, a nonspecific inhibitor is produced by an acceptor cell that has been armed with antigen-specific T suppressor factor and subsequently exposed to the corresponding antigen. FIGURE 1 illustrates the observations on T suppressor cells that depress delayed hypersensitivity to the NP (4-hydroxy-3-nitrophenylacetyl) and the arsanil groups.? The injection of haptenized lymphoid cells gave rise to antigendirected Ts (Ts, ) cells. These cells liberated an antigen-specific T suppressor factor (TSF). The cells or the factor then acted as an immunogen and induced the formation of Ts, (Ts) in the presence of more antigen. This Ts, is an anti-idiotypic suppressor cell; it is interesting to ask why the Ts, should act

Nonspecific inhibitor of contact sensitivity made by T-acceptor cells: triggering of T cells armed with antigen-specific T-suppressor factor (TsF) requires both occupancy of the major histocompatibility complex recognition site by soluble I-J product and cross-linking of the antigen recognition sites of the TsF.

The phenomenon of associative recognition, i.e., the recognition of antigen together with major histocompatibility complex products (MHC) was studied in a model system. T-acceptor cells armed with antigen-specific T-suppressor factor (TsF) released a nonspecific inhibitor of the transfer of contact sensitivity when exposed to antigen together with MHC. The MHC product occurred in a KCl extract of cells and behaved genetically and serologically as I-J. Cells armed with anti-picryl or anti-oxazolone TsF could be triggered by the corresponding bis-picryl-L-lysine and bis-oxazolone-L-lysine together with MHC. This suggested that cross-linking of antigen recognition sites on separate molecules of TsF might be required. To investigate this possibility the bifunctional mixed hapten N alpha-picryl-N epsilon-oxazolone-L-lysine, which is univalent with respect to the picryl and oxazolone haptenic groups, was synthesized. This triggered cells armed with a mixture of anti-picryl and anti-oxazolone TsF but not cells armed with either TsF alone. It was concluded that both occupancy of the I-J recognition site and the cross-linking of separate molecules of TsF was required for triggering. Moreover the hapten and the KCl extract could be given sequentially and in either order. This finding suggested that the triggering of the release of nonspecific inhibitor was due to the separate recognition of I-J and antigen and not to new antigenic determinants produced by their interaction.

Functional equivalence of cryptococcal and haptene-specific T suppressor factor (TsF). I. Picryl and oxazolone-specific TsF, which inhibit transfer of contact sensitivity, also inhibit phagocytosis by a subset of macrophages.

Monoclonal and conventional cryptococcal-specific T suppressor factors (TsF) (also called TsFmp) depress phagocytosis by a subset of macrophages, while picryl- and oxazolone-specific TsF depress the passive transfer of contact sensitivity. This paper shows that these haptene-specific TsF also inhibit phagocytosis by a subset of macrophages and, using this assay, that the anti-haptene TsF resemble the anti-cryptococcal TsF in five respects: (i) the need for reexposure to specific antigen to trigger the release of TsF; (ii) genetic restriction in action; (iii) possession of an antigen-binding site; (iv) expression of I-J determinants; and (v) inactivation by reduction and alkylation. Purification of the anti-picryl TsF by sequential affinity chromatography indicates that the inhibition of phagocytosis is due to the TsF itself and not to a TsF-antigen complex. The TsF inhibits phagocytosis by a direct action as macrophages treated with TsF and exposed to antigen do not release a second factor which inhibits phagocytosis. These results and those of the accompanying paper indicate that the anti-cryptococcal and anti-haptene TsF are functionally equivalent, antigen-specific suppressor factors.

Two-chain structure of T-suppressor factor: Antigen-specific T-suppressor factor occurs as a single molecule and as separate antigen-binding and I-J+ parts, both of which are required for biological activity

Antigen-specific T-suppressor factor (TsF), which acts at the expression stage of the contract sensitivity reaction, was produced by culturing the lymphoid cells of mice injected with picryl-sulphonic (trinitrobenzenesulphonic) acid and then painted with picryl chloride. Supernatant activity was found around 50-60 and 90 kDa on Sephadex gel filtration. The activity at 50-60 kDa was due to two separate (or readily separable) molecules, one antigen binding and the other bearing I-J determinants as shown by affinity chromatography on insolubilized antigen and anti-I-J. These two separate molecules were inactive alone but complemented each other and may be designated as the variable chain of TsF (TsFv) and the I-J+ chain. The use of gel filtration and sequential absorption of individual pools on anti-I-J antibody followed by antigen, together with a complementation assay, also showed a TSFv chain at 30-40 kDa and an I-J+ chain at 20-30 kDa. The higher-molecular-weight activity around 90 kDa was due to a single molecule which was both antigen binding and I-J+. This molecule dissociated on treatment with the reducing agent dithioerythritol followed by alkylation into two separate chains, one antigen binding and the other I-J+, both of which were required for activity. There was a requirement for genetic matching between the antigen-binding chain (TsFv) and the I-J+ chain for biological activity. These data support a two-chain model of TsF in which TsFv and the I-J+ chain occur as a single disulphide-bonded molecule around 90 kDa, or as two separate (or readily separable) chains of lower molecular weight which were inactive alone but complemented each other.

Control of suppressor cell activity: Autoanti-idiotype B cells produced by painting with picryl chloride inhibit the T-suppressor cell which blocks the efferent stage of contact sensitivity

Abstract This paper describes a B “suppressor of suppressor” cell which blocks the production or action of the T-suppressor cell, Ts-eff (cs), which acts at the efferent stage of the contact sensitivity reaction. Ts-eff (cs) occur in mice 7 days after injecting picrylsulfonic acid (PSA) and are assayed by their ability to block the passive transfer of contact sensitivity in a 24-hr experiment. These Ts-eff (cs) cannot be demonstrated in mice painted with picryl chloride and injected with PSA 8 days later. In fact, 8 days after painting mice contain B cells which prevent the appearance of Ts-eff (cs) following the injection of PSA. Moreover, the serum of mice 12 days after painting contains antibody which inactivates Ts-eff (cs). This antibody is anti-idiotypic as shown by its absorption to and elution from insolubilized mouse anti-picryl antibody and the lack of effect of absorption with insolubilized picryl groups. The antibody belongs to the IgG2a class and requires an intact Fc moiety for its action.

T suppressor factor activity is due to two separate molecules. The Lyt-1(-)2+I-J+ cells of mice primed with antigen make an antigen binding molecule which is only active when complemented by cofactor made by Lyt-1+2(-)I-J+ cells.

Mice primed with picrylsulfonic acid (PSA) and then painted on the skin with picryl chloride produce antigen-specific T suppressor factor (TsF). In contrast unpainted primed mice fail to produce active TsF. This is not due to the absence of the antigen binding part of TsF but to the absence of a cofactor. This cofactor is (a) antigen nonspecific and occurs in potassium chloride extract of normal spleen cells. It also occurs in the 24 hr supernatant of normal cells modified by haptenisation with picryl or the unrelated NP antigen (4-hydroxy-3-nitrophenylacetyl), and in preparations of conventional TsF (PSA/PCl) from painted PSA-primed mice; (b) bears I-J determinants; and (c) is produced by Lyt-1+2(-)I-J+ cells. The antigen binding molecule occurs alone in the supernatant of PSA-primed mice. It lacks I-J determinants and has a molecular weight around 35,000 and 75,000. It is produced by Lyt-1(-)2+I-J+ cells and is only active when complemented by cofactor. However, the complementation is genetically restricted and the restriction maps to the I-J subregion of the MHC.