Antigen-specific T contrasuppressor factor in cell-mediated immunity: interactions leading to eradication of the tolerant state

Interactions between a T cell-derived, antigen-specific, contrasuppressor factor (TcsF) and immune T cells that block the action of T suppressor factors and allow the transfer of cellular immunity into tolerant recipients are described. Immune T cells from contact-sensitized donors are capable of transferring specific immunity into normal recipients but not into animals rendered tolerant to the specific antigen. Brief exposure of the immune cells to the TcsF enables the effective transfer of immunity into such tolerant recipients. In addition, treated immune cells become resistant to subsequent exposure to T suppressor factor (capable of inhibiting transfer of immunity to normal recipients). A cyclophosphamide-sensitive, I-J+, Ly-2 T transducer cell is required in the immune donor cell population for contrasuppression to be induced by the TcsF plus specific antigen. These cells release an antigen-non-specific contrasuppressive factor capable of rendering immune targets, depleted of transducer cells, resistant to suppression (either by suppressor factor or in the tolerant recipient). The results indicate that contrasuppression in contact sensitivity is antigen specific and that the balance of suppression and contrasuppression determines tolerance vs responsiveness in this system. The symmetrical resemblance of the contrasuppressive interactions to those of suppression in contact sensitivity are discussed.     

Murine transfer factor. IV. Studies with genetically regulated immune responses

Transfer factor-containing dialysates from mice that were either high or low responders to GAT10, GLA5, or ovalbumin were assayed for their ability to transfer delayed hypersensitivity to murine recipients of either high or low responder phenotype. Dialysates from high responder strains contained transfer factor that would transfer delayed hypersensitivity to both high and low responder recipients. These transfers were not restricted by disparities at the MHC or Igh loci. Identically prepared materials from low responder donors contained little or no transfer factor activity and would not transfer delayed hypersensitivity to either high or low responder recipients. Thus, administration of transfer factor transfers the high responder phenotype to low responder recipients. The data also suggest that production of transfer factor is regulated by Ir genes but that the immunologic activities of transfer factor are not.     

Studies on tolerance induction in vitro. I. Production of immunologically tolerant rabbit spleen cells and the transfer of tolerance to untreated cells.

Tolerance was induced in rabbit spleen cells by incubation with solubilized T2 phage (S-T2)2 at 37degrees C. Spleen cells thus treated maintained normal responsiveness to an unrelated antigen, S-SP82. Transfer of tolerance was demonstrated in in vitro in that the addition of washed tolerant cells caused suppression of the response of untreated cells to an immunogenic dose of S-T2. Evidence is presented that this suppression is not due to the transfer of tolerogenic quantities of antigen. Spleen cell populations depleted of adherent cells were still capable of being made tolerant and of transferring tolerance.     

Transfer factors: identification of conserved sequences in transfer factor molecules

BACKGROUND: Transfer factors are small proteins that "transfer" the ability to express cell-mediated immunity from immune donors to non-immune recipients. We developed a process for purifying specific transfer factors to apparent homogeneity. This allowed us to separate individual transfer factors from mixtures containing several transfer factors and to demonstrate the antigen-specificity of transfer factors. Transfer factors have been shown to be an effective means for correction of deficient cellular immunity in patients with opportunistic infections, such as candidiasis or recurrent Herpes simplex and to provide prophylactic immunity against varicella-zoster in patients with acute leukemia. MATERIALS AND METHODS: Transfer factors of bovine and murine origin were purified by affinity chromatography and high performance liquid chromatography. Cyanogen bromide digests were sequenced. The properties of an apparently conserved sequence on expression of delayed-type hypersensitivity by transfer factor recipients were assessed. RESULTS: A novel amino acid sequence, LLYAQDL/VEDN, was identified in each of seven transfer factor preparations. These peptides would not transfer expression of delayed-type hypersensitivity to recipients, which indicates that they are not sufficient for expression of the specificity or immunological properties of native transfer factors. However, administration of the peptides to recipients of native transfer factors blocked expression of delayed-type hypersensitivity by the recipients. The peptides were not immunosuppressive. CONCLUSIONS: These findings suggest that the peptides may represent the portion of transfer factors that binds to the "target cells" for transfer factors. Identification of these cells will be helpful in defining the mechanisms of action of transfer factors.     

Quinpramine ameliorates rat experimental autoimmune neuritis and redistributes MHC class II molecules

Activation of inflammatory cells is central to the pathogenesis of autoimmune demyelinating diseases of the peripheral nervous system. The novel chimeric compound quinpramine--generated from imipramine and quinacrine--redistributes cholesterol rich membrane domains to intracellular compartments. We studied the immunological and clinical effects of quinpramine in myelin homogenate induced Lewis rat experimental autoimmune neuritis (EAN), a model system for acute human inflammatory neuropathies, such as the Guillain-Barré syndrome. EAN animals develop paresis of all limbs due to autoimmune inflammation of peripheral nerves. Quinpramine treatment ameliorated clinical disease severity of EAN and infiltration of macrophages into peripheral nerves. It reduced expression of MHC class II molecules on antigen presenting cells and antigen specific T cell proliferation both in vitro and in vivo. Quinpramine exerted its anti-proliferatory effect on antigen presenting cells, but not on responder T cells. Our data suggest that quinpramine represents a candidate pharmaceutical for inflammatory neuropathies.     

Frequency of RNA-RNA interaction in a model of the RNA World

The RNA World model for prebiotic evolution posits the selection of catalytic/template RNAs from random populations. The mechanisms by which these random populations could be generated de novo are unclear. Non-enzymatic and RNA-catalyzed nucleic acid polymerizations are poorly processive, which means that the resulting short-chain RNA population could contain only limited diversity. Nonreciprocal recombination of smaller RNAs provides an alternative mechanism for the assembly of larger species with concomitantly greater structural diversity; however, the frequency of any specific recombination event in a random RNA population is limited by the low probability of an encounter between any two given molecules. This low probability could be overcome if the molecules capable of productive recombination were redundant, with many nonhomologous but functionally equivalent RNAs being present in a random population. Here we report fluctuation experiments to estimate the redundancy of the set of RNAs in a population of random sequences that are capable of non-Watson-Crick interaction with another RNA. Parallel SELEX experiments showed that at least one in 10(6) random 20-mers binds to the P5.1 stem-loop of Bacillus subtilis RNase P RNA with affinities equal to that of its naturally occurring partner. This high frequency predicts that a single RNA in an RNA World would encounter multiple interacting RNAs within its lifetime, supporting recombination as a plausible mechanism for prebiotic RNA evolution. The large number of equivalent species implies that the selection of any single interacting species in the RNA World would be a contingent event, i.e., one resulting from historical accident.     

Induction of specific suppressor T cells in vitro.

We describe conditions for generating sheep red blood cell-specific suppressor T cells in Mishell-Dutton cultures. The production of specific suppressor cells is favored by increasing antigen dose in the initial culture but can be produced by transferring more cells when lower doses of antigen are used. Transfer of small numbers of cells cultured with low doses of antigen leads to a specific helper effect. Transfer of large numbers of educated cells leads to nonspecific suppression. Suppression can be effected by the effluent cells from nylon wool columns which do not make detectable PFC. A fraction of these cells become resistant to treatment with anti-T cell sera and complement after culture. The suppressor cells are radiation sensitive and must be able to synthesize protein to suppress. They take 2 to 3 days of education to reach maximum suppressive efficiency and will not suppress cultures if added 2 to 3 days after culture initiation. Their production is favored by the absence of mercaptoethanol, suggesting that the observed suppression is not "too much help". The ability to generate specific suppressor cells in vitro should be of great benefit in determining the factors that regulate their appearance in vivo.     

Nonrecirculating memory T lymphocytes in cellular resistance to infection

Acquired resistance of rats to intracellular infection with Listeria monocytogenes rests on the cooperation between sensitized mediator lymphocytes and effector macrophages. Large numbers of specific T lymphoblasts, capable of transferring resistance to recipients, appear in central lymph shortly (3–6 days) after subcutaneous infection of rats. In contrast, late-phase immunity is poorly transferrable with thoracic duct lymphocytes (TDL) despite high levels of specific resistance observed in spleen, liver, and testes of actively immunized animals. That late-phase immunity is mediated partly by resident, nonrecirculating T cells is attested to by the ineffective transfer of resistance from preinfected to normal partners of parabiotic rats. Transfer studies with thoracic duct cells and peritoneal cells from the stimulated and unstimulated peritoneal cavity seem to suggest that resident T cells mediating late-phase resistance are the progeny of lymphoblasts that extravasated during early phase. Assays measuring the proliferative response upon antigen stimulation in vitro support the concept of a gradual redistribution within the animal of memory T cells.     

Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F'' factor.

Filter matings between E. coli K-12 strains carrying an F'::Tn5,Tn9 factor with H. influenzae Rd strains gave rise to kanamycin-chloramphenicol-resistant H. influenzae strains at a frequency of approximately 10(-6). Transfer of the F' factor to H. influenzae was verified by expression of unselected markers in H. influenzae (lac+ or cotransfer of the nonselected antibiotic resistance), physical presence of a high-molecular-weight plasmid in recipient H. influenzae cells, and detection by Southern hybridization analysis of DNA sequences specific for the F' factor replication and partition functions in recipient H. influenzae cells. H. influenzae (F' Tn5,Tn9) strains were capable of transferring kanamycin and chloramphenicol resistances to other H. influenzae strains and were capable of mobilizing H. influenzae chromosomal markers at a low frequency. Insertion of a Tn5 element in the H. influenzae genome near the novobiocin resistance gene increased the frequency of transfer of novobiocin resistance about 30-fold. Transfer of other chromosomal markers also increased, although to a lesser extent, and ordered transfer of chromosomal markers could be demonstrated. Gene transfer was insensitive to DNase I, and transfer of chromosomal (but not F' factor) markers was dependent on the H. influenzae rec-1 and rec-2 gene functions.     

Surface molecules and cell interactions

Many of the cell surface molecules of lymphocytes or their precursors are expressed in an unpredictable way on a limited set of other cell types. This often seems to involve expression on lymphoid and brain cells. The Thy-1 antigen is in this category, being a major glycoprotein of murine neuronal cells, fibroblasts and thymocytes. Structural studies show that this molecule is homologous with immunoglobulin domains which are the structural sub-units of all immunoglobulin polypeptides. Thy-1 is the size of one immunoglobulin domain and its sequence is most homologous with variable regions of immunoglobulins.It is suggested that Thy-1 is one of a set of surface molecules concerned with triggering interactions between cells and that this is the primitive function of the immunoglobulin domain. Cell interactions could be mediated by domain-like structures and receptors for them in a way which parallels the triggering of immunological effector reactions by the interaction of receptors with immunoglobulin constant regions. If this is so then the structure seen in the immunoglobulin domain would have evolved along with the evolution of cell organisation. The genes specifying the cell interaction molecules could then have provided the genetic material for the evolution of antibody and histocompatibility antigen at the time of vertebrate emergence.     

Genetic functions and cell–cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis

Pheromone-inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1-5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell-cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.     

Cellular basis of genetic nonresponsiveness to egg white lysozyme

Utilizing radiation chimeras, we have investigated the cellular level at which the low immunological responsiveness to egg white lysozyme C57Bl/10 mice is expressed. Both NR----F1 (responder) and F1----NR combinations were assessed. The results demonstrate that C57Bl/10 bone marrow can give rise to hen egg white lysozyme responsive cells, but this response requires that the antigen be presented by cells derived from high responder animals.     

The influence of cyclosporin A on the development of actively induced and passively transferred experimental allergic encephalomyelitis

The immunosuppressive effects of cyclosporin A (CsA) were tested on actively induced and passively transferred experimental allergic encephalomyelitis (EAE). Actively induced EAE could be inhibited if CsA was administered per os at 25 mg/kg/day but not at 10 mg/kg/day. Passive transfer of clinical EAE occurred in all cell recipients including those fed CsA at either 25 or 50 mg/kg/day. Cyclosporin A could inhibit the development of transfer active cells in vitro and in vivo, however, inhibition of transfer active populations by CsA required the presence of CsA during the initial stage of cell response. If CsA was added to Con A-stimulated spleen cell cultures after a delay of 24 hr then these cells transferred clinical disease. Similarly, animals fed CsA concurrently with basic protein sensitization did not develop cell populations capable of transferring EAE. If CsA feeding commenced 2 or 4 days following sensitization all basic proteinsensitized animals still failed to develop EAE; however these latter groups of animals were a suitable source of cells capable of transferring some signs of clinical EAE.     

Immune response to the p-azobenzenearsonate (ABA)-GAT conjugate: role of I region genes in the selective activation of ABA-specific or GAT-specific T helper cells

Immunization of GAT non-responders with ABA-GAT leads to the activation of ABA-specific T cells. These hapten specific T cells are Lyt-1+2- helper cells capable of inducing anti-ABA antibody responses in vivo or B cell activation in vitro. However, their activation does not modify the GAT non-responder phenotype. Immunization of GAT responder mice with ABA-GAT activates GAT-specific T cells, which can help anti-ABA and anti-GAT antibody responses. Since the responder and non-responder strains used in these experiments differ only in the alleles present in the I region, the results suggest that the selective activation of hapten- or carrier-specific T cells is controlled by I region genes. Yet sensitization of the two strains with ABA-KLH or ABA-Tyr induces KLH-specific or ABA-specific T cells, respectively. This provides further evidence that the use of an immunogenic carrier prevents the expression of the hapten-specific T cell clones present in the repertoire of both responder and non-responder animals. Macrophages from responder animals pulsed with ABA-GAT can present ABA and GAT determinants to T cells. Thus, the absence of ABA-specific T cells in responders primed with ABA-GAT and their presence in GAT non-responders reflects a competition between hapten- and carrier-specific T cells and not an epitope selection by macrophages. We discuss the significance of the results in terms of Ir genes determining the self-plus-antigen-specific T cell repertoire rather than controlling antigen presentation by macrophages.     

Activities and characteristics of transfer factors

This report summarizes three components of our transfer factor research program. Several clinical studies have used oral administration of transfer factor containing materials. Sceptics have rejected these findings by assuming that the acidic and enzymatic environment of the gastrointestinal tract would destroy the factors. To further examine this issue, we have conducted dose-response studies of the delayed-type hypersensitivity reaction in mice that were given transfer factor either by gavage or subcutaneously. There were no difference in the responses that were related to the route of administration. We conclude that oral route of administration is efficacious and should be used when possible. We have also studied the effects of transfer factors on immune responses by recipients. The details of this research are presented in the paper by Dr. Alvarez-Thull. Briefly, the study showed that recipients of a specific transfer factor responded to the antigen for which the factor was specific by secreting gamma-IFN, but no other cytokines. The structures of transfer factor molecules are unknown. We have developed a process for isolating transfer factors in pure form and we have obtained preliminary data concerning amino acid sequences. Our goal is to obtain the complete primary structure of several transfer factor molecules.     

The role of H-2 in T cell recognition of Mls

The role of H-2 was evaluated in T cell recognition of Mls-encoded antigens during primary mixed lymphocyte responses (MLR). Mlsc was used as a stimulating determinant in MLR and its recognition by T cells was assessed by linear regression analysis under culture conditions in which (A x B)F1 responder cell number was the factor limiting total response. Results of such experiments indicated the presence of distinct (A x B)F1 responder T cell subpopulations capable of differentially recognizing the foreign Mls antigen in association with one or the other parental H-2 haplotype. These findings demonstrate that T cells do not recognize Mlsc products in isolation, but rather are restricted to recognition of Mlsc in the context of "self" H-2 determinants.     

Cell transfer studies in cyclophosphamide-induced tolerance

Thymectomized, irradiated adult CBA mice were restored with various combinations of bone marrow and thymus cells from nontolerant animals and from animals made tolerant to sheep erythrocytes or to hemocyanin with the drug cyclophosphamide. Mice reconstituted with tolerant marrow and thymus responded as well as those that received nontolerant cells. Thus it is concluded that the tolerant state of the transferred marrow and thymus cells is not a significant factor in the tolerant state of the recipient, and that antigenic diversity is restored in the interaction and proliferation of bone marrow and thymus cells that follow transfer.Thymectomized irradiated mice restored with thymocytes, in contrast to unoperated animals, require multiple antigen injections to demonstrate comparable immune response, but develop tolerance normally when treated with cyclophosphamide and antigen. Reconstitution with tolerant marrow and thymus cells resembles the recovery of immune responsiveness seen after lethal irradiation of tolerant mice; in both instances a complete breakdown of immunological tolerance is observed.     

Genetic restriction of delayed-type hypersensitivity to tuberculin in mice

An adoptive local transfer method has been used to study the immunological features and genetic restriction of cell interaction during the development of the delayed-type hypersensitivity (DTH) to tuberculin in mice. Peritoneal cells from the BCG-infected mice transfer the DTH to intact animals (into hind footpad) in both syngeneic and allogeneic donor-recipient combinations. Nonadherent cells (macrophage-deleted) transfer the reaction in syngeneic but not allogeneic combination. The use of H-2 recombinant mouse strains demonstrated that successful transfer of the DTH requires I-A subregion compatibility. Treatment of CBA cells with anti-Thy-1.2 antiserum abrogates the reaction transfer. These results indicate that antigen presentation to immune T-cells proliferating during DTH to tuberculin is mediated through the molecular products of the I-A subregion.     

MHC control of T lymphocyte-macrophage interactions

Identity at the major histocompatibility complex (MHC) of primed T cells and macrophages was essential for the development of a T cell proliferative response to Purified Protein Derivative of tuberculin (PPD) in the presence of macrophage-associated antigen and potential allogeneic effects were eliminated by the use of one-way fetal liver chimeras as a T cell source. By contrast, such MHC restriction could not be shown for the T cell—macrophage interaction when antigen was present in soluble form.It was found that the proliferative response of primed (responder × nonresponder) F₁ T cells to the Ir-gene controlled antigen, TNP-18 [Glu-Tyr-Lys (TNP) (Glu-Tyr-Ala)₅], could only be restored by responder macrophages with bound antigen, while both responder and nonresponder macrophages reconstituted the response to soluble TNP-18. Supernatants from cultured responder or nonresponder macrophages could at least partially replace viable macrophages in the latter case.These results argue for two distinct antigen presentation mechanisms, depending on the physical state of the antigen rather than its chemical nature: one involves recognition of antigen in association with MHC-coded determinants and shows H-2 restriction, while the other, mediated by soluble factors and antigen, does not.     

Model for immunologic testing of biomaterials]

Corrosion products and electric fields are capable of changing proteins to antigens, thus permitting the immunological system to identify the biomaterial as foreign. The reaction between corrosion products and a macro-molecule also leads to an antigen (carrier antigen), such as conformational changes of a macro-molecule, e.g. a protein, caused by the electric field at the implant surface (modified macro-molecule antigen). While the sensitivity to corrosion and the effectiveness of galvanic elements is measurable by electrochemical methods, suitable methods of determining the field strength in the vicinity of biomaterial surfaces are still unavailable. The influence of the double layer of uncoated and coated titanium surfaces on the conformation of proteins and their conversion to antigens are investigated with polyclonal antibodies capable of identifying the unchanged protein despite adsorption to the surface. 14C-marked Bovine Serum Albumin serves as a model protein. Determination of the total number of protein molecules adsorbed is effected via the detection of the emitted electrons. The quotient of the concentration of natural proteins to the concentration of adsorbed molecules gives the biocompatibility index, which is independent of the surface area, and gives an indication of the expected biocompatibility of the material. The results of the biological tests of titanium and two coating materials on titanium were confirmed in an animal experiment. It is possible that in the future immunological tests may replace experiments in animals.