The role of antigen recognition and suppressor cells in mice with oral tolerance to ovalbumin.

The induction of tolerance by feeding proteins may prevent potentially harmful delayed-type hypersensitivity (DTH) reactions to food antigens. Suppressor T cells (Ts) are present in mice with tolerance of systemic DTH after feeding ovalbumin (OVA) but, as other immunoregulatory mechanisms have also been described, the exact role of Ts in maintaining tolerance is not known. In this study, we have used the ability of native and denaturated OVA to cross-react at the level of helper/effector T cells, but not Ts, to re-examine the role of Ts in oral tolerance to OVA. Mice immunized with native OVA (nOVA) or denatured OVA (dOVA) in adjuvant had fully cross-reacting DTH to either nOVA or dOVA, but intravenous administration of antigen induced Ts which were specific for the appropriate form. Mice fed nOVA or dOVA had identical tolerance of systemic DTH to both forms of OVA, and feeding nOVA induced splenic Ts which suppressed the DTH response to both nOVA and dOVA. Splenic Ts could not be detected in mice fed dOVA. The results support the hypothesis that tolerance of systemic DTH in mice fed native proteins is due to Ts. Although, for the moment, there is no complementary evidence for a role for Ts in oral tolerance to denatured proteins, this study is consistent with the idea that Ts are the mechanism which normally prevent enteropathy due to DTH against dietary proteins. In addition, our study underlines the differences between orally and parenterally induced Ts and reinforces the view that fed proteins induce Ts after processing by the gut or its lymphoid accessory cells.     

Aberrancies in antigen-presenting cells and T cells in autoimmune thyroid disease. A role in faulty tolerance induction

Various thyrocyte, monocyte, macrophage, DC and T cell abnormalities exist in the animal models of spontaneously developing autoimmune thyroiditis and in patients with autoimmune thyroid disease. An aberrant interaction between such abnormal thyrocytes, abnormal professional antigen-presenting cells (APC) and abnormal T cells forms the basis for the atypical autoimmune reaction targeting thyroid antigens. In the atypical interaction more than one gene and various environmental factors are involved. The genetic and environmental factors must act together to induce full-blown disease. Although there is a general blueprint for the development of destructive autoimmune thyroiditis, thyrocyte and immune cell abnormalities differ between the various animal models and the various forms of autoimmune thyroid disease (either associated with type 1 diabetes, associated with bipolar disorder or not associated). This tells us that there are different etio-pathogenic forms of destructive autoimmune thyroiditis. Whether such heterogeneity is also the case for the etio-pathogenesis of Graves' disease remains unknown. Animal models of spontaneously developing Graves' disease would be helpful in unraveling this question. If indeed there are various etio-pathogenic routes in different patients that lead to destructive autoimmune thyroiditis, then tailor-made therapeutic approaches need to be carried out in attempts to correct the underlying immune abnormalities in individual patients or to prevent the development of destructive autoimmune thyroiditis in individuals at risk. While in some forms of destructive autoimmune thyroiditis (f.i. those associated with bipolar disorder) immune suppression should be the first choice of intervention, other forms (f.i. those associated with type 1 diabetes) may benefit from immune stimulation in certain pre-stages of the disease (to restore f.i. the faulty APC function characteristic of this condition). Obviously a more precise determination of the spectrum of cell-mediated immune abnormalities is required in individual cases of destructive autoimmune thyroiditis, before therapies that aim at correcting the immune abnormalities can be tested successfully.     

Genetic control of oral tolerance to ovalbumin in mice.

We have investigated the genetic basis of oral tolerance to OVA in a number of inbred mouse strains. Our results emphasise the efficiency of the oral route for inducing tolerance and provide evidence for both MHC and non-MHC linked control of oral tolerance.     

Suppressor T cells in low zone tolerance. I. Mode of action of suppressor cells.

Low zone tolerance (LZT) to bacteriophage fd seems to be a type of tolerance which is primarily caused by suppressor T cells. The aim of this paper is to analyze their mode of action. For the induction of antigen-specific suppressor cells in hydrocortisone pretreated CBA mice, we use tolerogenic and immunogenic doses of antigen. Suppressor activity can be demonstrated upon transfer of spleen cells into normal syngeneic mice. After immunization these animals are unable to produce IgG antibody against phage fd, whereas the IgM response is not suppressed; The half-life of transferred suppressor cells in nonimmunized animals is 5--6 weeks. The target of suppression are unprimed T helper cells, whereas primed helper cells cannot be blocked. T helper cells become "resistant" to suppression 18--36 h after contact with antigen. Differentiation from unprimed B into B memory cells is unaffected, yet under suppression conditions persisting B memory cells are blocked in IgG production. The experimental data are incorporated into a model of LZT.     

Sensitization of allo-specific T lymphocytes in vivo: role of antigen-presenting cells.

The migratory behavior of antigen-presenting cells was investigated in vivo. Purified murine splenic dendritic cells and splenic and peritoneal macrophages were labelled and injected subcutaneously in the hind foot-pads of mice and monitored for seven days. In the first 24 h, a small quantity of label was recovered from popliteal but not inguinal lymph nodes with radioactive (111In-oxine and 3H-uridine) but not fluorescent (1,1'-dioctadecyl 3,3,3'3'-tetramethylindocarbocyanine perchlorate and fluorescein isothiocyanate) labelling of the antigen-presenting cells. Chemical fixation of the injected antigen-presenting cells had no effect on the detection of label in the popliteal lymph nodes, suggesting that it was unlikely to be due to active cellular migration. Label recovery from hind feet declined with time over the seven day period and was independent of the label type. Essentially the same observations were made whether the antigen-presenting cells were syngeneic or allogeneic to the injected mice and irrespective of the type of antigen-presenting cell used. However, allogeneic antigen-presenting cells, which did not migrate to the draining lymph nodes, successfully primed T lymphocytes in these lymph nodes as shown by a secondary in vitro mixed leukocyte reaction. Again, chemical fixation of the injected antigen-presenting cells had no effect on their ability to prime allogeneic T lymphocytes in the draining lymph nodes. These experiments suggest that, during experimental allo-sensitization via the subcutaneous route, indirect priming of allogeneic T lymphocytes may be a dominant pathway.     

Epitopes associated with major histocompatibility complex (MHC) restriction site of T cells. IV. I-J epitopes on MHC-restricted cloned T cells

We studied the expression of an I-Jk epitope on class II-restricted cloned L3T4+ T cells established from H-2k, H-2b, F1 and semiallogeneic radiation bone marrow chimeras by the inhibition of antigen-induced T cell proliferation and in vitro secondary antibody response, and by the direct immunofluorescence with a monoclonal anti-I-Jk. Both I-Ak- and I-Ek-restricted T cells were shown to carry the identical I-Jk epitope regardless of their genotypic origins, antigen specificity, and helper or suppressor function. None of the I-Ab-restricted clones derived from similar animals showed the I-Jk epitope. This isomorphism, regardless of the restriction specificity for I-Ak or I-Ek, contradicts the idea that I-J is an idiotypic determinant on class II-restricted T cell antigen receptor (TcR). In fact, the I-Jk epitope was not comodulated with TcR/T3 complex when incubated with an anti-T3 antibody, indicating that I-J is a new isomorphic receptor for self different from TcR alpha/beta heterodimers.     

Suppressor T cells in low zone tolerance. II. Characterization of suppressor T and amplifier cells by physical and serological methods.

Suppressor cells involved in low zone tolerance to bacteriophage fd have been characterized by serological and physical methods. Suppressor T cells are peripheral T2 cells which are Ly-1-, Ly-2+ and Ia+. They are of high electrophoretic mobility, sediment 5 mm/h at 1 x g in a linear Ficoll 70 gradient, but are not restricted to a distinct band in a discontinuous bovine serum albumin gradient. Adherent phagocytic cells of low electrophoretic mobility can act as amplifier cells with a much shorter half-life than suppressor T cells.     

Modulation of oral tolerance to ovalbumin by cholera toxin and its B subunit.

Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally. Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline. Oral CT plus OVA also induced good serum IgG1 and IgA anti-OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses. Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the Th1 CD4+ T cell subset and with CT effect on B cells (induction of LPS-stimulated IgM+ B cells to undergo increased switch differentiation to IgG1- and IgA-secreting cells).     

The role of antigen-presenting cells in the regulation of allergen-specific T cell responses

Allergic reactions in atopic patients follow from a generalized enhanced polarization of Th cells, predominantly imposed by factors derived from antigen-presenting cells from a pathogen-stressed tissue; these sample information not only on antigen structures but also on the nature of the stress. Antigen-presenting cells of atopic individuals show aberrant characteristics which, through a highly interactive communication network, play an active role in aberrant Th-cell polarization. This generalized bias may follow from intrinsic abnormalities of antigen-presenting cells and also from a low degree of cross-regulation by micro-organisms.     

The role of small intestinal antigen-presenting cells in the induction of T-cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance.

The oral administration of soluble protein antigen results in profound immunological tolerance. However, the tissue location and function of antigen-presenting cells (APC) that stimulate this response remain unclear. We have hypothesized that the properties of cells presenting antigen to naive T cells within the gut are involved, and therefore gut APC should stimulate T-cell responses with different characteristics to those induced by other APC. To test this, we studied in vitro primary T-cell responses following presentation of soluble protein antigen by cells from the Peyer's patches (PPC) and lamina propria (LPC) of the murine small intestine and the spleen (SPLC). Each APC population stimulated antigen-specific proliferative responses with similar anamnestic characteristics; however, analysis of the cytokines produced revealed marked differences. Whereas SPLC stimulated the balanced production of T-helper type 1 (Th1) and Th2 cytokines, PPC induced a profile consistent with the provision of T-cell help for IgA production. Interestingly, presentation of antigen by LPC stimulated high levels of interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) in the absence of other cytokines [interleukin-2 (IL-2), IL-4, IL-5]. Evidence from analysis of cell activation and division within the cultures suggested that this profile may result from the preferential activation of CD8+ T cells by LPC; however, the lack of conventional CD4+ T-cell cytokines indicated a defect in the normal function of these cells. Adoptive transfer of antigen-pulsed LPC to syngeneic animals abrogated the induction of delayed-type hypersensitivity (DTH) responsiveness, which followed a subsequent conventional antigen challenge further suggesting a role for lamina propria APC in tolerance induction.     

Covalent coupling of palmitate to ovalbumin inhibits and blocks the induction of oral tolerance

Oral tolerance is a phenomenon that may occur in animals exposed to soluble antigens for the first time by the oral route. In the present study, we show that oral tolerance against ovalbumin (Ova) can be obtained after intragastric administration of the antigen in the presence of free residues of palmitate. On the other hand, oral tolerance induction is blocked when the residues of palmitate are covalently bound to the antigen (Ova‐palmitate conjugates). We have also noticed that oral administration of Ova‐palmitate conjugates can boost and/or prime experimental animals for Ova‐specific cellular and humoral systemic immune responses. Oral treatment with the conjugates also induces the production of local secretory immunoglobulin A (IgA) as measured in intestinal washes. Furthermore, Ova‐palmitate given orally can inhibit oral tolerance induction by naïve Ova.     

诱导人T细胞对猪抗原免疫耐受的体外实验研究

目的：探讨诱导人Ｔ细胞对猪抗原免疫耐受的状况。方法：通过建立人幼稚淋巴细胞与猪抗原递呈细胞混合培养模型,以淋巴细胞增殖实验及毒性实验,ＤＮＡ分析,淋巴细胞表面标志检测等手段,观测了幼稚淋巴细胞分化,发育过程中对异种负调细胞疫苗初次接触与再次刺激的免疫应答状况的变化。     

Suppressor T cells and the immune response to tumors

In this paper we have attempted to define the role of suppressor T cells in many well-defined murine tumor systems. We have knowingly omitted a blocking antibodies, suppressor B cells as mediators of tumor immunosuppression in various murine tumor systems; these have been well reviewed elsewhere. Also, we have focused on the importance of two different types of antigen-presenting cells in the induction and suppression of cell-mediated immunity and on some of the different modalities employed to inhibit Ts function. Finally, we have discussed the acquired immunodeficiency syndrome and the possible role of a defective helper pathway and enhanced suppressor pathway in its pathogenesis. We and others believe that the suppressor pathway is preferentially activated by tumor antigen(s) in the cases of many immunogenic murine tumors--possibly due to the release of tumor antigen(s) from tumor cells, their subsequent trafficking to specific areas of the spleen and other organs, and, ultimately, their presentation by certain APC to Ts. Ts may then act directly upon helper Lyt 1+2- T cells as these cells interact with tumor antigen(s) on I-A+ APC. Alternatively, if the effector pathway were somehow impaired--e.g., by ultraviolet radiation or a virus--then the suppressor pathway may be activated in an unregulated manner, often to the detriment of the host. Biochemical characterization of the tumor antigens that stimulate Ts generation and, presumably, tumor growth and definitive documentation of a role of APC in the processing and presentation of these tumor antigens to Ts need to be done. Then selective stimulation of the effector immune response, along with inhibition of the suppressor response, to tumor antigens with drugs, monoclonal antibodies, and soluble mediators or their analogues may be possible in the near future.     

Suppressor T cells which block the induction of cytotoxic T cells in vivo.

Mice pretreated with cyclophosphamide have an increased ability to produce anti-trinitrophenyl cytotoxic T cells after painting with the contact sensitizing agent picryl chloride. This could be abrogated by injecting normal cells or cells from mice exposed to trinitrophenyl derivatives at the time of painting. If the injection of cells was delayed until 1 day after painting specificity could be demonstrated. Normal cells and cells from mice injected with dinitrobenzene sulphonate were ineffective whereas cells from donors injected with picryl sulphonic acid were inhibitory. Inhibitory cells were also shown in mice painted with picryl chloride, particularly after adult thymectomy. Using this system it was found that cells from picryl chloride but not oxazolone painted mice were inhibitory when injected 1 day after painting the recipients. The suppressor cells from the mice injected with picryl sulphonic acid and from the mice painted with picryl chloride were shown to be cyclophosphamide sensitive T cells and were not affected by adult thymectomy. These properties have helped to classify the suppressor cells induced by trinitrophenyl derivatives.     

The response of T cells to soluble protein antigens and fixed antigen-presenting cells.

A polyclonal population of antigen-specific T cells has been used to study the presentation of a soluble protein antigen (ovalbumin) using a B-cell line (A.20) as antigen-presenting cells. We find that glutaraldehyde-fixed antigen-presenting cells will present undegraded antigen to this polyclonal population of T cells. T-cell proliferation is observed only if an exogenous source of T-cell growth factor is added to the cultures. These findings are compared to the results obtained using antigen-specific T-cell clones or T-cell hybridomas and provide further evidence to suggest that degradation of antigen is not essential for the presentation of antigen to T cells.     

Immunological responses to fed protein antigens in mice. I. Reversal of oral tolerance to ovalbumin by cyclophosphamide

Feeding ovalbumin over a wide range of doses is known to reduce subsequent systemic immune responses to parenteral immunization. In the present study, we have fed mice 2 mg and 25 mg ovalbumin (OVA) 2 weeks before systemic immunization and followed the resulting humoral antibody and cell-mediated immune (CMI) responses. The results indicate that while 25 mg OVA will reduce subsequent IgM, IgG and CMI responses to OVA, feeding 2 mg OVA will only suppress CMI responses and to a lesser extent the IgM response. Furthermore, the tolerant state induced by feeding 25 mg OVA was only partially prevented by 100 mg/kg cyclophosphamide (CY) while the suppressed CMI after feeding 2 mg OVA was completely blocked by CY pretreatment. These findings suggest that the humoral and cell-mediated limbs of the immune response may be controlled by different regulatory systems after feeding antigen, and that activation of these systems is dependent on the dose of oral antigen use. In addition, the results are in agreement with our previous finding that CY pretreatment will allow the development of CMI in the gut and gut-associated lymphoid tissue (GALT) after oral OVA and suggest that this phenomenon is related to breakdown of oral tolerance induction.     

Complementary role of CD4+CD25+ regulatory T cells and TGF-beta in oral tolerance

CD4(+)CD25(+) regulatory T cells are thought to be generated in the periphery as well as in the thymus. We sought to determine the roles played by CD4(+)CD25(+) T cells and transforming growth factor-beta (TGF-beta) in the induction and maintenance of tolerance generated by oral antigens in BALB/c mice. We found that oral administration of a high dose of ovalbumin (OVA) suppressed OVA-specific proliferation and antibody production in BALB/c mice depleted of CD25(+) cells. In contrast, the unresponsiveness induced by lower doses of OVA was only partially blocked by CD25 depletion prior to feeding. Depletion of CD4(+)CD25(+) cells after mice were orally tolerized did not reverse the tolerant status, indicating that these cells were not required to maintain the established tolerance. Furthermore, the induction of oral tolerance was not hampered by the administration of TGF-beta-neutralizing antibodies. However, in mice depleted of CD25(+) cells, anti-TGF-beta-neutralizing antibodies blocked the induction of tolerance, regardless of whether the mice followed the high- or low-dose regimens of oral OVA. CD25 depletion together with TGF-beta neutralization led the expansion of OVA-specific CD4 T cells against the subsequent antigen challenge, and each treatment alone did not. Our findings indicate that CD4(+)CD25(+) T cells and TGF-beta play a complementary role in the induction of oral tolerance, at least in part, by regulating the expansion of antigen-specific CD4 T cells.