Enterically induced immunologic tolerance. I. Induction of suppressor T lymphoyctes by intragastric administration of soluble proteins.

Specific immune unresponsiveness was induced in inbred mice (BDF1) by the administration of soluble ovalbumin (OVA) by gastric intubation. Anti-hapten (DNP) responses likewise were specifically diminished when animals were fed autologous carrier (OVA or keyhole limpet hemocyanin). Adoptive transfer of spleen cells demonstrated that the tolerant state could be maintained in irradiated recipient mice, and specific anergy could be transferred to normal recipient animals. Adoptive suppression was mediated by T lymphocytes, as demonstrated by nylon wool fractionation and susceptibility of the cells to anti-Thy 1.2 and complement. Transferred B cells had neither suppressive nor augmentative effects. Enteric administration of OVA also specifically diminished antigen-induced DNA synthesis of primed lymph node T cells, although suppressor cells were not identified in the lymph nodes per se.     

The regulation of resistance to Schistosoma mansoni by auto-anti-idiotypic immunity. III. An analysis of effects on epitopic recognition, idiotypic expression, and anti-idiotypic reactivity at the clonal level.

Auto-anti-idiotypic mechanisms can regulate the protective immune response against Schistosoma mansoni. Anti-idiotypic responses were stimulated by immunization of mice either with nonspecifically induced lymphoblasts, produced with Con A, or with Ag-induced lymphoblasts bearing specific idiotypic receptors. The effect of the induced anti-idiotypic response upon clonotypic cellular reactivity was assessed in vitro through the suppression of antigen-mediated blast transformation by cloned T cells and in vivo by suppression of resistance to S. mansoni and delayed-type hypersensitivity responses against specific Ag. Differential regulation of humoral immune responses was studied at the levels of specific epitopic recognition, the expression of specific Id, and the production of anti-idiotypic responses directed against mAb bearing specific Id. Anti-idiotypic sensitization resulted in variable (10 to 90%) suppression of the immune response to discrete antigenic epitopes, the expression of specific idiotypic phenotypes, and anti-idiotypic, antiparatopic responses against T cell clonotypes and antibody idiotypic phenotypes. In vitro admixture and in vivo challenge studies resulted in consonant differential suppression. Thus idiotypic regulation can mold the fine specificities of the protective immune response to S. mansoni at the clonal level and may provide an approach to optimize the expression and assessment of resistance.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

The effects of handling on antibody production, mitogen responses, spleen cell number, and lymphocyte subpopulations

The effects of a simple psychosocial manipulation, handling, on immune function in BALB/c.ByJ mice were studied. Handling for two minutes/day prior to immunization was shown to be associated with a decreased primary IgG antibody response to the protein antigen keyhole limpet hemocyanin. Handling was also associated with decreased T cell proliferative responses to Concanavalin A. No reliable changes in spleen cell number or lymphocyte subsets were found. These findings demonstrate that simply picking up an animal results in modulation of the immune response. These data are also of interest for their obvious methodological implications.     

Carrier-specific immune memory to a thymus-independent antigen in congenitally athymic mice.

Immune memory to the DNP epitope coupled to a nonmitogenic thymus-independent carrier, Ficoll, was demonstrated in congenitally athymic outbred Swiss mice. Strong IgM and modest IgG components of this memory were detected. Moreover, this memory was carrier specific since it was elicitable only when DNP-Ficoll primed mice were challenged with DNP-Ficoll and not when similarly primed mice were challenged with DNP coupled to pneumococcal polysaccharide or to keyhole limpet hemocyanin. Ficoll primed mice also demonstrated a memory response when challenged with DNP-Ficoll. These findings indicate that a non-T cell, presumably a B cell, is capable of recognizing the carrier epitopes of this thymus-independent hapten-carrier complex. Unlike their athymic counterparts, euthymic mice of the same genetic background failed to demonstrate the IgM component of this memory, but they did demonstrate modest carrier-specific IgG memory. These results strongly suggest that suppressor T cells are either directly or indirectly important in regulating the IgM memory response of these mice to DNP-Ficoll. Indirect regulation could possibly occur via an antibody-mediated specific immune suppression mechanism.     

Antibody response against a possible arthritogenic epitope on human type II collagen induced by anti-idiotypic antibody

We reported the presence of three distinct epitopes commonly present on murine and human type II collagen (CII), observed using mAb. To investigate the possible involvement of these epitopes in collagen-induced arthritis, we raised rabbit anti-idiotypic antibodies that may bear the internal image of these epitopes. Anti-idiotypic antibodies developed against three anti-CII mAb designated as 1-5, 2-14, and 2-15 were demonstrated to recognize idiotype expressed on Ag-binding site (paratope) of their related mAb. Anti-CII antibody response specific for a given epitope could be induced in DBA/1J mice upon immunization with anti-idiotypic antibodies coupled to keyhole limpet hemocyanin. Anti-idiotypic antibody to 1-5 antibody in particular could stimulate all DBA/1J mice for production of anti-CII antibody possessing Ag specificity and idiotype similar to those of 1-5 antibody. Although the mice immunized with anti-1-5 antibody alone did not develop arthritis, they did show a much more enhanced antibody response against a given epitope than did control mice non-treated with anti-idiotypic antibody upon the subsequent immunization with human CII. Some of the mice immunized with anti-1-5 antibody and challenged with human CII developed arthritis, whereas the control mice did not. These findings strongly suggest that a common epitope recognized by 1-5 antibody might be involved in the induction of arthritis.     

Resistance in murine schistosomiasis is contingent on activated IL-2 receptor-bearing L3T4+ lymphocytes, negatively regulated by Lyt-2+ cells, and uninfluenced by the presence of IL-4

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. C57BL/6 mice were depleted in vivo of L3T4+, Lyt-1+, Lyt-2+, IL-2R+ cells, or IL-4 by administration of appropriate mAb. Resistance and various correlative parameters of the immune response were studied in normal, depleted, and congenitally athymic mice. Depletion of T lymphocytes by anti-L3T4 or anti-IL-2R mAb reduced the development and expression of resistance, IgG2a and IgE antibody formation, and delayed type hypersensitivity reactivity against schistosome Ag. Depletion with anti-IL-4 antibody led to profound suppression of IgE-eosinophil-mediated antibody-dependent cell-mediated cytotoxicity and passive cutaneous anaphylaxis responses against the parasite and no effect on IgG2a antibody, Ag-mediated blast transformation, or resistance. Depletion of Lyt-2+ cells produced augmented development and expression of resistance and an increase in the immunological parameters of anti-schistosome reactivity. These studies suggest that protective immunity to S. mansoni in mice, induced by irradiated cercariae, is dependent on L3T4+, IL-2R+ lymphocytes and negatively regulated by Lyt-2+ cells. IL-4 does not appear to be essential for the development of resistance but is essential for the IgE response to the parasite.     

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.     

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.     

Effects of 60-Hz electric fields on specific humoral and cellular components of the immune system

We evaluated humoral and cellular functions of the immune system of Swiss-Webster mice exposed to 60-Hz electric fields at 100 kV/m. No significant differences were observed in primary antibody response to keyhole limpet hemocyanin (precipitating antibody levels) between exposed (30 or 60 days) and control mice, nor were there significant changes in mitogen-stimulation response of spleen cells from mice similarly exposed for 90 or 150 days when compared to sham-exposed animals.     

Influenza-specific suppression: contribution of major viral proteins to the generation and function of T suppressor cells

Suppressor cells were generated in BALB/c mice by two sequential injections of PR8 influenza virus (A/Puerto Rico/8/34[H1N1]) and were tested for their ability to inhibit proliferative cellular responses towards multiple viral and nonviral antigens. In this way, suppression specific to PR8 as compared with purified protein derivative (PPD) and keyhole limpet hemocyanin (KLH) antigenic responses was illustrated. Experiments involving adoptive transfer of suppression to naive hosts with subfractionated lymphocyte populations demonstrated that the suppressors were Lyt-2+ T cells. Two major questions were addressed with this system. First, a determination was made of which anti-viral protein proliferative responses were affected by the PR8-induced T suppressor (Ts) cells. Ts cells were found to inhibit proliferating cells with specificities for isolated hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix (M) antigens. Second, experiments were conducted to analyze the viral proteins contributing to the induction of PR8-specific Ts cells. Inoculations with either isolated HA or a combination of M + NP proteins induced T suppression specific to proliferative responses towards PR8. These experiments illustrate the contribution of external (HA and NA) as well as internal (M + NP) viral proteins to Ts cell generation and function.     

MHC class II+ exosomes in plasma suppress inflammation in an antigen-specific and Fas ligand/Fas-dependent manner

Exosomes are 50- to 100-nm vesicles that are formed within the late endocytic compartment and released from a variety of cell types. Previously, we demonstrated that exosomes derived from dendritic cells transduced with adenoviral vectors expressing IL-10, IL-4, or Fas ligand (FasL) produce anti-inflammatory exosomes able to reduce inflammation in a murine paw delayed-type hypersensitivity model, suppress the onset on murine collagen-induced arthritis, and reduce the severity of established collagen-induce arthritis. In this study, we examined the ability of endogenous, blood-borne exosomes to regulate the immune response. Exosomes isolated from plasma of mice immunized to keyhole limpet hemocyanin, but not from naive or OVA-immunized mice, were able to suppress the keyhole limpet hemocyanin-specific delayed-type hypersensitivity inflammatory response. The anti-inflammatory effect was mediated by MHC class II(+) plasma exosomes that were also FasL(+) and CD11b(+), but CD11c(-). Moreover, the anti-inflammatory effect of the MHC class II(+) plasma-derived exosomes was, in part, dependent upon the presence of FasL in the exosomes and Fas in the recipient mouse. These results suggest that exosomes in the plasma, produced by MHC class II(+) and CD11b(+) cells, have the ability to suppress the immune response in an Ag-specific manner in part through a Fas/FasL-dependent manner.     

The cellular and humoral immune response to Schistosoma mansoni infections in inbred rats. V. Prerequisite mechanisms for the development of optimal protective immunity.

The mechanisms whereby natural infection or artificial immunization lead to the development of optimal protective immunity against reinfection by homologous Schistosoma mansoni were investigated. Animals, exposed to normal or irradiated cercariae, demonstrated strong resistance to reinfection by S. mansoni. They also developed a population of T lymphocytes, which could adoptively transfer resistance in vivo and specifically interact with immunogen in vitro. Subsequently these animals produced antibody capable of adoptively transferring resistance and possessing a variety of antischistosomal activities. Animals exposed to soluble cercarial immunogen demonstrated moderate resistance upon reexposure. They failed to produce a significant population of sensitized T lymphocytes. Moreover, although these animals produced unimpaired levels of cytotoxic, complement-fixing, and hemagglutinating antibody, their sera did not adoptively transfer resistance. Adult worm and egg immunogens were even less effective than cercarial immunogen in stimulating resistance. In addition, these membrane-derived immunogens directly stimulated B-lymphocyte blastogenesis in vitro. These studies suggest that the optimal production of protective immunity requires the stimulation of T-dependent mechanisms by stage-specific immunogens. This stimulation is produced more effectively by the use of natural infection or irradiated cercariae than by the use of soluble schistosome immunogens which may be working through relatively T-independent mechanisms.     

Production of idiotypic and anti-idiotypic antibodies by BALB/c mice in response to immunizations with glucagon, vasopressin, or insulin: supporting evidence for the network concept

After immunizations with glucagon or vasopressin, either conjugated to keyhole limpet hemocyanin or adsorbed to polyvinylpyrrolidone, both anti-hormone and anti-receptor activities were detectable in the serum of injected mice. Anti-hormone activity was identified by ELISA techniques; anti-receptor activity, by determining the ability of serum samples to compete with labeled hormone for glucagon or vasopressin receptors on rat liver plasma membranes. Anti-receptor activity appeared only after the peak anti-hormone response to each immunogen had been established, and required intensive immunizations (six to nine monthly injections). The presence of anti-idiotypic antibodies in serum samples containing glucagon or vasopressin anti-receptor activity was confirmed by demonstrating selective binding of such samples to corresponding rabbit idiotypic antibodies. Serum from mice immunized with insulin also contained anti-hormone activity, as determined by ELISA, and anti-receptor activity, as determined by noting insulin-mimicking properties in stimulating glucose transport in rat adipocytes. The anti-insulin receptor activity developed after only one boost with the hormone. These results are consistent with Jerne's network hypothesis in that the glucagon, vasopressin, and insulin anti-receptor activity may be attributed to antibodies produced in mice as part of an idiotypic-anti-idiotypic network.     

Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate

The capsule of Bacillus anthracis, a polymer of gamma-D-glutamic acid, functions as a virulence determinant and is a poor immunogen. In this study we show that antibodies reactive with the B. anthracis capsule can be elicited in mice by immunization with a conjugate consisting of a synthetic gamma-D-glutamic acid nonamer peptide (gamma-D-glu9) covalently coupled to keyhole limpet hemocyanin. The serum response to gamma-D-glu9 was comprised primarily of IgG antibodies that recognized an epitope requiring a minimum of four gamma-linked D-glutamic acid residues. Antibodies to (gamma-D-glu9) bound to the surface of encapsulated B. anthracis cells and mediated opsonophagoctosis. These findings suggest that anti-capsular antibodies could mediate the clearance of vegetative B. anthracis cells in vivo. Thus, inclusion of an immunogenic capsular component as well as protective antigen in new anthrax vaccines would generate immune responses targeting both the bacteremic and toxigenic aspects of anthrax infection and thus may increase protective efficacy.     

Monoclonal antibodies to an artificial immunogen, specifically demonstrating phosphotyrosine-containing proteins

The importance of protein phosphorylation at tyrosyl hydroxy groups in the control of cell proliferation has recently been established. For identification of tyrosine-phosphorylated proteins, monoclonal antibodies (Mabs) against artificial immunogens containing O-phosphotyrosine (pTyr) or tripeptide pTyr-Gly-Gly as haptens were generated; the haptens were coupled to carrier proteins (bovine serum albumin, human immunoglobulin, keyhole limpet hemocyanin). After immunization of mice with pTyr coupled to keyhole limpet hemocyanin, Mabs were generated which were highly specific for pTyr and did not cross-react with O-phosphoserine, O-phosphothreonine, tyrosine or nucleoside-5'-monophosphates. The Mabs specifically react with tyrosinephosphorylated proteins in the Rous sarcoma virus-transformed rat XC-cell.     

Low dose ultraviolet B-irradiated Langerhans cells preferentially activate CD4+ cells of the T helper 2 subset

UVB radiation distorts the Ag-presenting function of epidermal Langerhans cells (LC); this has been shown for the presentation of soluble Ag to primed T cells in vitro and for the initiation of delayed-type hypersensitivity in vivo, such as contact hypersensitivity (CH). Previous work has also demonstrated UVB-induced suppression of CH to be mediated ultimately by T cells. Two subsets of CD4+ Th cells, Th1 and Th2, have been identified, based on their cytokine production and functional activities. In particular, Th1 mediate delayed-type hypersensitivity, whereas Th2 do not. To investigate whether the perturbation of LC function induced by UVB radiation leads to a differential activation of these subsets of CD4+ cells, we examined the capacity of unirradiated and irradiated (200 J/m2) APC from adult BALB/c mice to present keyhole limpet hemocyanin to Ag-specific, H2d-restricted Th1 and Th2 cell lines. Four sources of APC were utilized: epidermal cells (EC), flow microfluorometry-purified Ia+ EC (LC), flow microfluorometry-purified Ia- EC, and splenic adherent cells (SAC). Unirradiated EC, LC, and SAC, but not Ia-EC, presented keyhole limpet hemocyanin to both Th1 and Th2. Irradiated EC and LC lost their ability to stimulate Th1, but retained fully their capacity to stimulate Th2. On the other hand, irradiated SAC were unable to induce proliferation of either Th1 or Th2. These findings indicate that suppression of CH mediated by UVB-irradiated LC may result from an alteration of the ratio and/or activity of Th1 and Th2 cells normally generated during the induction of such responses.     

Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.     

Tumor cell lysate-pulsed dendritic cells are more effective than TCR Id protein vaccines for active immunotherapy of T cell lymphoma

TCR Id protein conjugated to keyhole limpet hemocyanin (KLH) (TCR Id:KLH) and injected with a chemical adjuvant (QS-21) induces a protective, Id-specific immune response against the murine T cell lymphoma, C6VL. However, Id-based immunotherapy of C6VL has not demonstrated therapeutic efficacy in tumor-bearing mice. We report here that C6VL lysate-pulsed dendritic cells (C6VL-DC) vaccines display enhanced efficacy in both the prevention and the therapy of T cell lymphoma compared with TCR Id:KLH with QS-21 vaccines. C6VL-DC vaccines stimulated potent tumor-specific immunity that protected mice against lethal challenge with C6VL and significantly enhanced the survival of tumor-bearing mice. Tumor-specific proliferation and secretion of IFN-gamma indicative of a Th1-type immune response were observed upon ex vivo stimulation of vaccine-primed lymph node cells. Adoptive transfer of immune T cell-enriched lymphocytes was sufficient to protect naive recipients from lethal tumor challenge. Furthermore, CD8(+) T cells were absolutely required for tumor protection. Although C6VL-DC and control vaccines stimulated low levels of tumor-specific Ab production in mice, Ab levels did not correlate with the protective ability of the vaccine. Thus, tumor cell lysate-pulsed DC vaccines appear to be an effective approach to generate potent T cell-mediated immune responses against T cell malignancies without requiring identification of tumor-specific Ags or patient-specific Id protein expression.     

Mice with Th2-biased immune systems accept orthotopic corneal allografts placed in "high risk" eyes

CD4+ T cells of the Th1 type play a central role in acute rejection of solid tissue grafts, including orthotopic corneal allografts. Th1 cells, which mediate delayed hypersensitivity, are the polar opposites of CD4+ Th2 cells, and the latter cells cross-regulate Th1 cells through the unique pattern of cytokines they secrete. As such, Th2 cells may have a useful role to play in preventing rejection of corneal allografts. To test this possibility, the immune systems of adult mice were biased toward Th2 responses by immunization with keyhole limpet hemocyanin plus IFA. When immunized subsequently with either OVA or allogeneic corneal tissue, these mice acquired Ag-specific primed T cells of the Th2 type. More important, allogeneic corneas grafted into neovascularized eyes of Th2-biased mice experienced significantly enhanced survival. To demonstrate that enhanced survival was promoted by donor-specific Th2 cells, lymphoid cells from keyhole limpet hemocyanin-immune mice bearing healthy corneal allografts suppressed orthotopic corneal allograft rejection when adoptively transferred into naive, syngeneic recipients. We conclude that acceptance of corneal allografts in neovascularized mouse eyes can be significantly enhanced by biasing the recipient immune system toward Th2 responses.