Hansruedy Ramseier

In Vitro Inhibition of Cellular Immune Responses by Benzodiazepines and Pk 11195 Effects on Mitogen-and Alloantigen-driven Lymphocyte Proliferation and on IL-1, IL-2 Synthesis and IL-2 Receptor Expression

In vitro mitogen-driven lymphocyte proliferation tests (Con A, LPS) on murine lymph node and spleen cells revealed inhibition of T and B cell stimulation by different benzodiazepines and by PK 11195, with IC50 values in the low micromolar range. T cell responses as a consequence of recognition of alloantigens, as measured in mixed lymphocyte cultures (MLC), were affected in an analogous way. In all systems, agonists at peripheral type benzodiazepine receptors (Ro 5-4864 and the non-benzodiazepine compound PK 11195) and diazepam which acts on both, central and peripheral type benzodiazepine receptors, were most potent; clonazepam, a central type agonist, proved about half as active. The central type antagonist Ro 15-1788 failed to antagonize the action of diazepam and clonazepam. Variations among cells from several congenic strains of mice were modest. Cytotoxicity could not be made responsible for drug effects. The most susceptible stage of mitogen-triggered T and B lymphocyte proliferation was found to be at incipience. Radioresistant, adherent spleen cells, upon LPS-stimulation formed only small amounts of the cytokine IL-1. Its release was affected only at very high drug concentrations. Similar small amounts of IL-1 were generated during MLC; in this case, the drugs were about 10 times less potent than in mitogen-induced proliferation assays. Peripheral agonists were more active on IL-1 synthesis. Spleen cells stimulated with Con A and cultivated with the highest concentration of diazepam and clonazepam formed markedly greater amounts of IL-2 than those cultivated in medium, while at this concentration PK 11195 allowed no formation of the lymphokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to t cell receptors and to histocompatibility antigens.

Abstract The injection of 10 6 parental strain spleen cells into adult F1 hybrid mice resulted in the formation of two serum activities: anti-receptor antibodies and alloantibodies. To demonstrate the respective roles of T and B cells in the elicitation of these serum activities, parental strain lymphoid cell suspensions containing varying proportions of T and B cells, or consisting only of T or B cells, were employed as inocula for F1 mice. The results indicated that the injection of pure T cells led to the formation of antireceptor antibodies only, while the injection of pure B cells resulted exclusively in formation of alloantibodies, suggesting that anti-receptor antibodies were structures elicited by T cell receptors. Alloantibodies appear to be formed as a consequence of the interaction of B cell receptors with alloantigen.

Activation of T and B thymus cells to recognize histocompatibility antigens

Abstract Lethally irradiated (A × CBA) F 1 or (A × C57BL/6) F 1 mice were injected with 10 7 A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F 1 hybrid spleen cells) into corresponding unirradiated F 1 hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F 1 mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F 1 hosts not containing the antigens against which activation had been directed.

Studies on antireceptor antibody and on alloantibody formation

The injection of F1 hybrid spleen cells into lethally irradiated parental strain recipients resulted in the formation of antisera containing antireceptor antibody but no alloantibody activity. The results demonstrated radioresistance of the immunogenic property of T-cell receptors for alloantigens and confirmed earlier conclusions on the origin of antireceptor antibody-forming cell populations. The system employed invited investigation into a possible cooperation between F1 B and F1 T lymphocytes in the formation of anti-RS antibodies in unirradiated and in lethally irradiated parental strain hosts. In the absence of F1 T cells, F1 B cells were incapable of forming these antibodies and vice versa. Semiallogeneic synergism in which parental T cells were to cooperate with F1B cells could not be observed. Addition to F1B inoculum of T lymphocytes from syngeneic, from allogeneic, and from semiallogeneic sources before injection into parental recipients also gave results that excluded all but a syngeneic T-B cell collaboration. The production of antibodies to parental T-cell receptors, therefore, seems to require recognition of allogeneic receptor by both T and B lymphocytes.

Studies on antireceptor antibody and on alloantibody formation. I. Kinetics of antibody formation in semiallogeneic and in allogeneic immunizations.

Semiallogeneic and allogeneic immunization protocols were used to clarify the origin of two antibody activities: (a) those directed at alloantigens and (b) those directed at receptors for alloantigens. In a first variant of semiallogeneic immunization, parental strain spleen cells were injected into F1 hybrid hosts where they could not be rejected; both antireceptor antibody and alloantibody activities appeared, persisting for over 10 weeks. When the host was irradiated, no antireceptor antibody was formed, indicating the F1 origin of this activity. In the second variant of semiallogeneic immunization, F1 spleen cells were injected into parental hosts. Here the antireceptor antibody, while reaching high titers, even in hosts presensitized to antigens of the donor, soon disappeared, presumably due to the rejection of inoculated cells. It was concluded that in both variants of semiallogeneic situations, antireceptor antibodies were formed by F1 cells, whereas alloantibodies were formed by parental cells. In allogeneic immunizations, four activities were found in serum —two directed at alloantigens (one set from each partner) and two directed at receptors for alloantigens (one set from each partner) —indicating that lymphoid cells of any origin can function as producers of antibodies to alloreceptors, in strict analogy to their potential as producers of antibody to alloantigens.

Reconstitution of primary mixed-lymphocyte culture reactions inhibited with class I antibodies by a product of histoincompatible spleen cells

Primary MLC reactions with spleen cells from mice of conventional inbred or congenic origin and incompatible at the entire H-2 or at K- or D-regions could be inhibited by pretreating stimulator but not responder cells with mAb to Class I MHC antigens. Inhibited MLC reactions, initiated in the presence of a product (SEPIR) present in supernatants after short-term cultivations of one-way histoincompatible spleen cells of the same haplotype as MLC cells, could be restored specifically to normal activities. It is suggested that the reconstituting factor represents a product of Class I antigen recognition capable of compensating the prevented recognition of these antigens by MLC responder cells. SEPIR was ineffective in restoring Class II antigen-inhibited MLC responses. Because the product might be formed spontaneously under normal MLC conditions, it had no effect when added to uninhibited MLC cells. Conceivably due to neutralization, SEPIR was also incapable of restoring MLC responses conducted in the continuous presence of Class I mAb. Inhibition of MLC responses and reconstitution with SEPIR depended on the effectiveness of Class I antigen inhibition.

Anti-idiotypic antibodies induce formation of anti-H-2 sera and of idiotypes.

Alloantisera could be induced in parental strain recipients either by means of alloantigen (skin allografting) or by injecting anti-idiotypic (anti-T cell receptor) sera. The resulting sera displayed activity against H-2 antigens; they also contained soluble isiotypic structures. With these sera anti-idiotypic sera could be provoked in F1 hybrid hosts. If antibody cycles initiated with alloantigen were carried through four runs, specificity appeared to be preserved and titers obtained corresponded to those found in conventionally raised anti-idiotypic sera and in alloantisera.

Characterization of a product of histoincompatible spleen cells restoring class I-inhibited mixed-lymphocyte culture reactions

Conditions leading to the formation of a recognition product (SEPIR) capable of restoring MLC responses inhibited by treating stimulator cells with mAb to Class I antigens have been investigated. SEPIR has been found to be present in supernatants of immunological one-way interactions of histoincompatible spleen lymphocytes from naive mice following cultivation for a few hours. It is active in relatively high dilutions. Similarities with cytokines of IL-1, IL-2, or interferon character could not be revealed. Formation of the heat-labile principle is governed by immunologically specific reactions and involves recognition of Class I (but not Class II) HMC antigens by T cells of Lyt-2 phenotype. B cells and Lyt-1+ cells failed to induce formation of the product. SEPIR appears to be a complex of T-cell receptors for Class I antigens with these antigens. The data indicate that in conventionally induced fully incompatible MLC responses a similar product might be formed.

Early Recognition of Transplantation Antigens by T Lymphocytes

In crude supernates of interactions of parental strain T spleen cells with H-2 antigen-bearing F1 hybrid spleen cells, a product (soluble early product of immune recognition, SEPIR) can be found which enhances mixed leukocyte culture (MLC) reactions set up with suboptimal cell concentrations (SMLC). Supernates of parental T cells reacting with F1 spleen cell also produce this factor. The product ameliorates SMLC proliferative responses specifically since identical antigenic specificities have to be recognized in both SEPIR formation and in test SMLC. Further evidence for SEPIR being an immunologically specific product is documented by the finding that spleen cells from mice made specifically tolerant at birth cannot respond to the tolerogen with SEPIR formation, but are fully reactive towards unrelated H-2 antigens. No responses to syngeneic or autologous antigens could be discerned. Characterization of allorecognition resulting in SEPIR formation has revealed that spleen cells of Lyt 23 phenotype recognize H-2K/D-encoded antigens. Lyt 1 cells appear to be incapable of forming the early product and H-2I-determined antigens fail to incite a response. Reactivity to class I antigens was amenable to specific inhibition by monoclonal alloantibodies.In crude supernates of interactions of parental strain T spleen cells with H-2 antigen-bearing F1 hybrid spleen cells, a product (soluble early product of immune recognition, SEPIR) can be found which enhances mixed leukocyte culture (MLC) reactions set up with suboptimal cell concentrations (SMLC). Supernates of parental T cells reacting with F1 spleen cells also produce this factor. The product ameliorates SMLC proliferative responses specifically since identical antigenic specificities have to be recognized in both SEPIR formation and in test SMLC. Further evidence for SEPIR being an immunologically specific product is documented by the finding that spleen cells from mice made specifically tolerant at birth cannot respond to the tolerogen with SEPIR formation, but are fully reactive towards unrelated H-2 antigens. No responses to syngeneic or autologous antigens could be discerned. Characterization of allorecognition resulting in SEPIR formation has revealed that spleen cells of Lyt 23 phenotype recognize H-2K/D-encoded antigens. Lyt 1 cells appear to be incapable of forming the early product and H-2I-determined antigens fail to incite a response. Reactivity to class I antigens was amenable to specific inhibition by monoclonal alloantibodies.

Transplantation Tolerance at the T-Lymphocyte Receptor Level II.

CBA T lymphocytes deprived temporarily of receptors for alloantigens A[RS(A)] cultivated in vitro for 30 h with anti-receptor antibody-forming (AxCBA)F1 spleen cells were capable of resynthesizing RS(A) if primed F1 cells exceeded parental T cells by a factor of 25 or less, but not if the excess was 50-fold or more. This indicated that resynthesis of CBA T-cell RS(A) was successful if primed F1 cells formed insufficient amounts of anti-CBA T-cell RS(A) antibody. Abortive or successful receptor resynthesis was measured by two parameters, (a) reappearing RS(A) formed PAR together with A alloantigens of (AxCBA)F1 spleen cells and (b) budding receptors bound anti-receptor antibody. CBA B lymphocutes did not interfere with these reactions. A search for putative T suppressor cells in the F1 cell population was unsuccessful. PAR formation and anti--RS antibody consumption by reappearing receptors differed temporally: receptors forming PAR were present after a delay lasting 8 h; receptor structures fixed anti-RS antibody as early as 5 h after being cultivated. With due caution, these results might reflect processes operating in maintenance of transplantation tolerance, suggesting that this condition is a serum-mediated suppression of long duration. The suppression would encompass continued neutralisation of receptors for the alloantigen to be tolerated by anti-T-cell receptor antibody formed by the F1 chimeric cells within an animal with acquired transplantation tolerance.