Tolerogenic Doses Research Articles

Effects of Peptide-Induced Immune Tolerance on Murine Lupus.

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

CD5-dependent CK2 activation pathway regulates T cell activation and threshold for anergy (143.27)

Abstract We have reported that a major biological activity of CD5 is to regulate the survival of activated T cells, a property mediated by the CD5-dependent CK2 activation pathway. In order to address the physiological role of the CD5-dependent CK2 activation pathway in T cell activation, the generation of Th effector cells, and autoimmunity, we generated by gene targeting a mouse (CD5ΔCK2BD) that expressed CD5 lacking the ability to bind and activate CK2. CD4 and CD8 T cells from CD5ΔCK2BD mice exhibited enhanced AICD and hyporeactivity to antigen receptor stimulation compared to T cells from CD5WT and CD5-/- mice. The ability to mobilize calcium and upregulate CD69 were greatly enhanced in CD5-/- T cells but not in CD5WT T cells or CD5ΔCK2BD T cells, indicating that the CD5-dependent CK2 activation pathway did not regulate TCR-proximal signaling pathways. CD5-/- mice developed attenuated experimental autoimmune encephalomyelitis (EAE) when immunized with 150 μg MOG, yet the disease in CD5ΔCK2BD mice was comparable to that seen in CD5WT mice. In contrast, immunization with tolerogenic doses of MOG resulted in attenuated EAE in CD5WT mice but not in CD5-/- and CD5ΔCK2BD mice. The results indicated that loss of the CD5-dependent CK2 pathway made T cells refractory to induction of anergy, a prediction that was recapitulated in in vitro recall assays. Our results reveal a novel role for CD5 in setting thresholds for anergy in T cells. Supported by grants from the NIH and NMSS.

T cell-mediated oral tolerance is intact in germ-free mice

Commensal enteric bacteria stimulate innate immune cells and increase numbers of lamina propria and mesenteric lymph node (MLN) T and B lymphocytes. However, the influence of luminal bacteria on acquired immune function is not understood fully. We investigated the effects of intestinal bacterial colonization on T cell tolerogenic responses to oral antigen compared to systemic immunization. Lymphocytes specific for ovalbumin-T cell receptor (OVA-TCR Tg(+)) were transplanted into germ-free (GF) or specific pathogen-free (SPF) BALB/c mice. Recipient mice were fed OVA or immunized subcutaneously with OVA peptide (323-339) in complete Freund's adjuvant (CFA). Although the efficiency of transfer was less in GF recipients, similar proportions of cells from draining peripheral lymph node (LN) or MLN were proliferating 3-4 days later in vivo in GF and SPF mice. In separate experiments, mice were fed tolerogenic doses of OVA and then challenged with an immunogenic dose of OVA 4 days later. Ten days after immunization, lymphocytes were restimulated with OVA in vitro to assess antigen-specific proliferative responses. At both high and low doses of OVA, cells from both SPF and GF mice fed OVA prior to immunization had decreased proliferation compared to cells from control SPF or GF mice. In addition, secretion of interferon (IFN)-gamma and interleukin (IL)-10 by OVA-TCR Tg(+) lymphocytes was reduced in both SPF and GF mice fed OVA compared to control SPF or GF mice. Unlike previous reports indicating defective humoral responses to oral antigen in GF mice, our results indicate that commensal enteric bacteria do not enhance the induction of acquired, antigen-specific T cell tolerance to oral OVA.

Short-term treatment with mycophenolic acid and tacrolimus is tolerogenic for INS-1 cell clone transplantation and the deleterious effects of the drugs are limited: in vivo and in vitro studies

One of the major requirements for a successful and life-lasting organ transplant is the access to safe, least toxic and permanent tolerance-inducing drugs. In this study we wished to evaluate the effects of tolerogenic doses of the immunosuppressive drugs mycophenolic acid (MPA) and tacrolimus (Tac) on clonal beta-cell lines, both in vivo and in vitro. Here we demonstrate that combined administration of low-dose MPA and Tac for 23 days induced permanent tolerance in an allogeneic beta-cell line transplant in Wistar rat liver through the portal vein. This short-term treatment of tolerogenic doses of the two drugs was deleterious to the survival of the transplanted cells but a small percentage of the cells could resist the effect and become fully active when the drugs were removed. The surviving cells, retrieved from growth in vivo, did not exhibit increased resistance in comparison to the original cells when tested in vitro at two glucose concentrations, 10 and 20 mM. The presence of a small percentage of resistant cells at the two glucose concentrations was also detected in the in vitro study after a continuous 8-day treatment demonstrating that the in vivo resistance was not related to micro-environmental protection but possibly to a phenotypic cell state that is yet to be determined.

Ligation of Toll-Like Receptor 5 (TLR5) by Flagellin Overcomes Antigen-Specific CD4+ T Cell Tolerance Via Inhibition of IL10 and Induction of IL12 in Antigen Presenting Cells (APCs).

The immune system is very effective in fighting infections but seems not to be as efficient in recognizing and destroying cancer cells. In the cancer setting, tumor antigen uptake and presentation by APCs to antigen-specific T-cells often occurs in the absence of inflammation resulting therefore in tolerance induction. It is plausible therefore that by converting APCs from a non-inflammatory to an inflammatory phenotype through ligation of TLRs we may well overcome immune tolerance and tip the balance towards productive tumor antigen-specific T-cell responses. Among all the TLR-ligands identified to date, flagellin is the only one with a strictly proteinic nature, characteristic that make it a suitable candidate for cloning and transfection into tumor cells to generate novel tumor cell based vaccines. In this study, we first evaluated whether treatment with purified flagellin could prevent tolerance induction in vivo. Naïve CD4+ T-cells (2.5x106) specific for a MHC class II-restricted epitope of influenza hemagglutinin (HA) were adoptively transferred intravenously into BALB/c mice, 24 hours after mice were given either a tolerogenic dose of HA-peptide (200 mg), or a combination of this high dose of peptide together with Flagellin (10 mg I.V.). Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses towards the cognate antigen evaluated in vitro. As expected clonotypic T-cells isolated from animals treated with high dose peptide were fully tolerant, in sharp contrast with those isolated from flagellin treated animals that displayed normal responses in terms of cytokine production and proliferation. Surprisingly, this preservation of T-cell function following in vivo treatment with flagellin was not observed when animals were treated with high dose HA-peptide in the presence of the TLR4 ligand, LPS. To better understand the mechanism(s) by which flagellin, and not LPS, preserved the responsiveness of antigen-specific CD4+ T-cells to cognate antigen presented by APCs, we assessed the phenotypic characteristics and the cytokine profile of macrophages and DCs treated in vitro with these TLRs ligands. Although LPS-treated APCs produce higher levels of IL-12, relative to flagellin-treated APCs, the production of this pro-inflammatory cytokine was accompanied by a parallel induction of the anti-inflammatory cytokine, IL-10. Interestingly, flagellin-treated APCs produced IL-12 but were unable to produce IL-10. This effect was dependent on ligation of TLR5, since it was not observed when RAW264.7 cells -which lack TLR5- were treated with flagellin. In vivo studies further confirmed our observations since IL-10 was not detected in the serum of animals treated with flagellin, but it was present in significant amounts in LPS-treated animals. This inhibitory effect of flagellin on IL-10 production was seen even when APCs were stimulated in vitro with strong inducers of IL-10. Given the above properties of flagellin, we generated two novel approaches to be used in the formulation of tumor cell based vaccines: 1) A bystander cell line transfected with the fliC gene from Salmonella typhimurim for flagellin expression (B78H1-Flagellin) and 2) Microspheres beads coated with flagellin. Both vaccination strategies are being currently studied in the in vivo and in vitro settings

Anatomical and cellular basis of immunity and tolerance in the intestine.

Anatomical and cellular basis of immunity and tolerance in the intestine.

Induction of local innate immune responses and modulation of antigen uptake as mechanisms underlying the mucosal adjuvant properties of immune stimulating complexes (ISCOMS)

Lipophilic immune stimulating complexes (ISCOMS) containing the Quil A adjuvant can induce local and systemic immune responses against orally delivered protein antigens. Here, we have examined the possibility that ISCOMS act by modulating local innate immune responses and antigen uptake in the intestine. Feeding ISCOMS to mice induced recruitment of dendritic cells (DCs), activated macrophages (mφ) and lymphocytes into the mesenteric lymph node (MLN), as well as recruitment of macrophages and B cells into the PP. Feeding ISCOMS also enhanced the absorption into circulation of a test feed of ovalbumin (OVA), with accelerated kinetics and increased peak levels. Serum taken 1 h after feeding a tolerogenic dose of OVA to mice given ISCOMS orally did not induce the tolerance of systemic delayed type hypersensitivity (DTH) and antibody responses found in mice receiving serum from donors fed OVA alone. Thus, ISCOMS may increase the immunogenicity of intestinal antigen via a combination of effects on antigen uptake and on local accessory cells.

Suppression of Specific and Bystander IgE Responses in a Mouse Model of Oral Sensitization to β-Lactoglobulin

Background: Mechanisms of systemic IgE suppression by oral tolerance have been extensively studied, but less is known about oral tolerance induction in mice challenged at mucosal sites. We have previously shown in systemically challenged mice that high-dose tolerance suppressed specific but not bystander IgE. In an attempt to mimic oral tolerance in food-allergic patients, we have investigated how IgE suppression could be induced in mice sensitized orally against β-lactoglobulin (BLG). Methods: Mice were immunized orally against BLG using cholera toxin as adjuvant. Before oral sensitization, mice were administered milk whey proteins, either in the form of a single high-dose gavage, or by prolonged ad libitum administration of various doses. Results: Orally sensitized mice mounted a BLG-specific IgE response. In contrast to systemically challenged mice, a single high-dose gavage of whey protein given prior to the onset of oral sensitization resulted in the suppression of both specific and bystander IgE. When mice were fed moderate to low doses of milk whey proteins daily ad libitum in the drinking water during 3 weeks prior to oral sensitization, all doses effectively suppressed antigen-specific IgE. However, bystander IgE suppression was observed only at the lowest doses. When mice were tolerized during 4 days instead of 3 weeks, IgE titers remained unchanged. Conclusions: In orally sensitized mice, bystander IgE suppression depended on the dose of tolerogen, but also on its mode of administration. Mucosally induced IgE responses were suppressed by a mechanism that was distinct from that operating in the periphery.

Regulatory and effector CD4 T cells in nonobese diabetic mice recognize overlapping determinants on glutamic acid decarboxylase and use distinct V beta genes.

The 524--543 region of glutamic acid decarboxylase (GAD65), GAD65(524--543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524--543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-A(g7)-restricted determinants present in the GAD65(524--543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR V beta gene use. CD4(+) T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530--543 (p530); however, T cells to the overlapping determinant 524-538 (p524) dominate the response only after immunization with GAD65(524--543). All p530-responsive T cells used the V beta 4 gene, whereas the V beta 12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524--543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGF beta upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524--543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.

T cell receptor complementarity determining region 3 length analysis reveals the absence of a characteristic public T cell repertoire in neonatal tolerance. The response in the "tolerant" mouse within the residual repertoire is quantitatively similar but qualitatively different.

All adult BALB/c mice immunized with hen egg white lysozyme (HEL) or its dominant determinant, peptide (p)106-116, mount a T cell response using a "public" Vbeta8.2Jbeta1.5 T cell clone. Neonatal exposure to tolerance-inducing doses of antigen can drastically diminish responsiveness in the draining lymph nodes but not in the spleens of animals challenged as adults with the cognate antigen. To determine the role of T cell deletion or anergy within the mechanisms of observed neonatal "tolerance," we treated neonatal BALB/c mice with HEL and directly followed the characteristic public clone using complementarity determining region 3 length T cell repertoire analysis. Our results confirm that despite intraperitoneal injection of neonates with a high dose of HEL emulsified in incomplete Freund's adjuvant, a strong splenic proliferative response to HEL was observed upon recall. However, the adult splenic T cell response of these neonatally treated mice lacked the usual Vbeta8.2Jbeta1.5 public clone characteristic of HEL-primed BALB/c mice. After challenge with HEL-complete Freund's adjuvant as adults, immunoglobulin (Ig)G2a isotype antibody was drastically reduced, and IgG1 was found to be the predominant anti-HEL IgG isotype expressed, indicating a deviation of cytokine response toward T helper type 2. 5-wk-old mice, nasally instilled with tolerogenic doses of HEL p106-116, also showed significant inhibition of this public T cell expansion. These results demonstrate that during neonatal and adult nasal tolerance induction, deletion/anergy removes the public clone, exposing a response of similar specificity but that is characterized by the T helper type 2 phenotype and a splenic residence.

Differential Control of Neonatal Tolerance by Antigen Dose versus Extended Exposure and Adjuvant

Ig-PLP1, an immunoglobulin (Ig) chimera carrying the encephalitogenic proteolipid protein (PLP) sequence 139–151 (PLP1), induces neonatal tolerance in mice and confers resistance to experimental allergic encephalomyelitis (EAE) without the need for incomplete Freund's adjuvant (IFA). The mechanism underlying such tolerance involves organ-specific T cell regulation characterized by lymph node deviation and an unusual IFNγ-dependent splenic anergy. This form of T cell modulation may prove useful for prevention of autoimmunity. However, since the neonatal period is susceptible to regulation, further investigations are necessary to define parameters required to establish regimens suitable for optimal protection against disease. Therefore, studies were carried out to investigate the effect that IFA, the dose of Ig-PLP1, and the number of Ig-PLP1 injections might have on Ig-PLP1-mediated neonatal tolerance and protection against disease. Herein it is reported that as little as 1 μg of Ig-PLP1 supported IFNγ-dependent splenic anergy but lymph node deviation and protection against disease strengthened as the dose of tolerogen increased. However, when a two-injection regimen was applied, resistance to disease was observed but the mechanism manifested proliferative and cytokine unresponsiveness in both lymphoid organs. Furthermore, the use of IFA along with Ig-PLP1 yielded a suppressive mechanism similar to that of the two-injection regimen. Therefore, the dose of Ig-PLP1 displays a quantitative influence, while the number of injections of Ig-PLP1 and the presence of IFA rather drive qualitative influences on such tolerance.

Normal Induction of Oral Tolerance in the Absence of a Functional IL-12-Dependent IFN-γ Signaling Pathway

AbstractThere is considerable evidence that regulatory cytokines play an important role in mediating the systemic tolerance found after oral administration of protein Ags. Although most existing work has focused on cytokines such as IL-4, IL-10, and TGF-β, recent evidence from TCR transgenic systems suggests that the induction of oral tolerance is accompanied by priming of Ag-specific IFN-γ production. IFN-γ has also been implicated as a mediator of T cell tolerance in other models in vivo and in vitro, including that induced by aerosol administration of protein. We show here that feeding tolerogenic doses of OVA primes for IFN-γ production in the spleen of mice with a normal T cell repertoire. However, depleting IFN-γ at the time of feeding OVA had no effect on the induction of tolerance. In addition, tolerance was induced normally in both IFN-γ receptor knockout (IFN-γR−/−) and IL-12 p40 knockout (IL-12−/−) mice. This was the case for all components of the systemic immune response and also with a variety of feeding protocols, including those believed to induce distinct regulatory mechanisms. We conclude that IL-12-dependent IFN-γ-mediated regulation does not play an essential role in oral tolerance.

All or none peripheral tolerance induction in H-Y antigen-specific TCR transgenic mice

In in vivo tolerance induction, the dose of tolerogen injected is generally linearly correlated to and length of tolerance induced. Small, medium and large doses are related to no, partial and long-term tolerance, respectively. However, even with injection of substantially large doses of tolerogen, the length of tolerance induced varies over a wide range. Most of the recipients can still reject donor grafts. In this study, it is shown that the linear dose-response can be altered into an all or nothing response in a H-Y antigen-specific TCR transgenic (Tg) mouse model. In thymectomized female Tg mice, injection of 3, 30 and 100 × 10 6 male spleen cells was correlated to no, partial and massive deletion of Tg (α Tβ T) CD8 cells, respectively. When the thymectomized Tg mice were injected with 9 × 10 6 T cell-enriched (T+) male cells, one half of the recipients showed no deletion of α Tβ t cells, and in the other half massive deletion occurred. In complete correlation with deletion, all male skin grafts were rejected in the undeleted group as PBS-injected controls, whereas with massive deletion they were indefinitely tolerized. Thus, partial deletion and partial tolerance can be avoided. Injection of 18 × 10 6 male T+ cells induced long-term tolerance in all the recipients. The all or none T cell deletion and long-term tolerance induction has not only significant implications in understanding the mechanism of peripheral tolerance induction, but also in tolerance induction in transplantation, gene therapy and the prevention and treatment of autoimmune diseases.

Presentation of cryptococcal capsular polysaccharide (GXM) on activated antigen-presenting cells inhibits the T-suppressor response and enhances delayed-type hypersensitivity and survival.

A hallmark of infection with Cryptococcus neoformans is depression of the immune system characterized by poor inflammatory responses and loss of delayed-type hypersensitivity (DTH) and antibody responses. T-suppressor cell (Ts) responses, elicited by the capsular polysaccharide (GXM) of the organism, are known to develop during infection. This study was undertaken to develop a method to inhibit the anti-GXM Ts response and thereby study the influence of the Ts response on immune responsiveness and survival in cryptococcosis. Antigen-presenting cells (APC), elicited with complete Freund's adjuvant (CFA), were treated in vitro with GXM (GXM-APC). The GXM-APC were injected intravenously into normal mice. These mice were resistant to induction of anti-GXM Ts cells when soluble GXM was administered in tolerogenic doses or when animals were infected with C. neoformans. Inhibition of the anti-GXM Ts response was specific to GXM as levan-APC did not inhibit induction of anti-GXM Ts cells. Inhibition of the anti-GXM Ts response could not be attributed to increased clearance of GXM due to induction of anti-GXM antibodies or other mechanisms. Anti-cryptococcal DTH responses were lost in mice by the second week of infection. However, treatment with GXM-APC, but not levan-APC, allowed mice to maintain their DTH response. GXM-APC pretreatment enhanced survival of infected mice compared with mice pretreated with levan-APC. These results show that GXM-APC induces immune responses that inhibit the induction of Ts responses and enhances DTH responses in infected mice. These responses correlate with enhanced survival after cryptococcal infection.

Levels of peripheral T cell tolerance induced by different doses of tolerogen.

Antigen-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. A liver-specific inducible promoter was used to drive the expression of the major histocompatibility complex antigen Kb in transgenic mice. Minute amounts of Kb, expressed exclusively on hepatocytes, induced tolerance by partial down-regulation of the T cell receptor (TCR) on the self-reactive CD8+ cells. Contact of these tolerant T cells with high concentrations of Kb after induction led to complete down-regulation of TCR. Thus, tolerant T cells are susceptible to further tolerogenic signals and reach different levels of tolerance depending on antigen dose.

Effect of captopril treatment on allograft survival and induction of unresponsiveness in heart-transplanted ATG-treated rats

The angiotensin-converting enzyme inhibitor captopril has been suggested to have an immunomodulatory effect both in clinical and experimental studies. To further investigate this possible effect in organ transplantation, captopril was given to inbred rats before transplantation of an allogeneic heart. Captopril was found to prolong graft survival significantly compared to untreated controls. In rats treated with a tolerogenic dose of ATG, additional captopril administration tended to increase the proportion of rats with long-term functioning grafts, but this did not reach statistical significance. It is concluded that captopril displays an immunosuppressive effect that seems to be weak, since no augmentation of ATG-induced transplantation unresponsiveness was seen.

Oral Induction of Immunological Tolerance to Chromium in the Guinea Pig

Metal alloys used in dentistry may elicit adverse side-effects. Contact allergic reactions to metals released from such alloys are among the most frequently encountered problems. In an earlier study, we observed that oral contacts with nickel or chromium salts did not sensitize, but rather decreased the risk of subsequent sensitization to these metals. In the present study, we focused on chromium allergy and extended our earlier observations by further dose-response studies. In addition, we compared different chromium valencies as to their potential oral tolerogenic effects. Development of immunological tolerance in chromium-fed guinea pigs was demonstrated by their inability to develop chromium hypersensitivity after a subsequent immunization attempt. For these studies, the techniques of immunization and skin testing were first improved. One feeding with a high dose of K2Cr2O7 (containing hexavalent chromium) was effective in full tolerance induction. In contrast, trivalent chromium (CrCl3) induced a distinctly lower degree of tolerance, whereas metallic chromium powder was not detectably tolerogenic after a limited number of feedings. Dose-frequency-response studies with K2Cr2O7 showed that full tolerance could also be induced by an increase in the number of feedings with sub-optimal tolerogenic doses. The present results therefore support our hypothesis that long-lasting oral contact with chromium-releasing metal alloys may ultimately result in strong immune tolerance to this metal in subjects without previous skin contact with it. This view is further supported by recent insights into the unique tolerogenicity of oral, as compared with gastro-intestinal, allergenic contacts.

Resistance to experimental autoimmune thyroiditis: L3T4 + cells as mediators of both thyroglobulin-activated and TSH-induced suppression

Mechanisms suppressive to induction of murine experimental autoimmune thyroiditis (EAT) can be activated by pretreatment with tolerogenic doses of mouse thyroglobulin (MTg) or prior TSH infusion to raise circulatory MTg levels. MTg-activated suppressor T cells (Ts), shown earlier to be Thy-1 + and probably I-J +, were further characterized by in vivo administration of paired rat monoclonal antibodies to distinct epitopes on the L3T4 or Lyt-2 molecule, either on the day of, or subsequent to, initiation of the tolerogenic regimes. The cells required at the time of MTg pretreatment were L3T4 +, Lyt-2 − and low anti-L3T4 doses had no effect on their activation. The cells that mediated the strong MTg-induced resistance following pretreatment were also L3T4 +; their suppressor function could only be abrogated by depletion of L3T4 +, but not Lyt-2 +, cells. Injection of cyclophosphamide (20–100 mg/kg) either prior to EAT induction or after Ts activation did not affect the severity of disease. Similarly, the suppressor state evoked by TSH infusion could only be abrogated by anti-L3T4 treatment. These findings indicate that both MTg-activated and TSH-induced suppression are mediated by L3T4 + cells. We hypothesize that MTg-specific Ts are present in normal, EAT-susceptible mice in low numbers to contribute to the maintenance of self-tolerance and that they are stimulated by increased levels of circulatory MTg to expand/differentiate and mediate the marked resistance to EAT induction.

Effects of early antigen exposure through lactation on later specific antibody responses in mice.

This study characterized totally the effects of early Ag exposure by the suckling route on later specific antibody responses. When mother mice of BALB/c or C57BL/6 strains were injected with deaggregated human gamma-globulin (HGG) immediately after delivery, total amounts of HGG in sera of offspring increased until 2 wk of age. The catabolism of transferred HGG was extremely slow and the half-life was about 3 wk in both strains. Hence, small amounts of Ag in mothers, 0.5 micrograms in C57BL/6 and 50 micrograms in BALB/c, could tolerize their offspring effectively. As these were minimum tolerogenic doses, the strain difference in ease of tolerance induction is apparent already during suckling. The study on timing dependent effects of HGG-specific antiserum on tolerance induction by mothers given 50 micrograms HGG demonstrated that the tolerance is achieved within the 1st wk of lactation in C57BL/6 offspring, but not in BALB/c offspring, and the restoration from the tolerance needs more than 6 wk under circumstances, supposedly, without free Ag. Whereas the tolerance was induced in a dose-dependent manner in each class of antibody, the dissociation of tolerant states between IgM, IgG, and IgE antibody classes was found in C57BL/6 offspring. It is interesting that C57BL/6 offspring were sensitized weakly, but significantly, by mothers given subtolerogenic doses. However, this was not apparent in BALB/c. Thus, the Ag dose and the animal strain are related closely to the consequences of this Ag exposure. The aging of suckling mice within the first 2 wk of life or immunomodulators administered early in life did not seriously affect the consequences. Studies on a cellular basis showed that the tolerance is caused by the selective defect in helper T cell function and the suppressor cell activity is not associated with the mechanisms. This contrasts with other models of oral tolerance.

Immunopathology and male infertility.

Barriers between the male's immune system and his germ cells have been presumed. However, they turn out to be imperfect, which suggests that autoimmune attack may be prevented in part by chronic exposure of T lymphocytes to tolerogenic doses of “foreign” germ cell antigens. Breakdowns in such active immunoregulation may account for some cases of unexplained infertility.