Opportunistic Autoimmune Disorders Potentiated by Immune-Checkpoint Inhibitors Anti-CTLA-4 and Anti-PD-1.

To improve the efficacy of immunotherapy for cancer and autoimmune diseases, recent ongoing and completed clinical trials have focused on specific targets to redirect the immune network toward eradicating a variety of tumors and ameliorating the self-destructive process. In a previous review, both systemic immunomodulators and monoclonal antibodies (mAbs), anti-CTLA-4, and anti-CD52, were discussed regarding therapeutics and autoimmune sequelae, as well as predisposing factors known to exacerbate immune-related adverse events (irAEs). This review will focus on immune-checkpoint inhibitors, and the data from most clinical trials involve blockade with anti-CTLA-4 such as ipilimumab. However, despite the mild to severe irAEs observed with ipilimumab in ~60% of patients, overall survival (OS) averaged ~22-25% at 3-5 years. To boost OS, other mAbs targeting programed death-1 and its ligand are undergoing clinical trials as monotherapy or dual therapy with anti-CTLA-4. Therapeutic combinations may generate different spectrum of opportunistic autoimmune disorders. To simulate clinical scenarios, we have applied regulatory T cell perturbation to murine models combined to examine the balance between thyroid autoimmunity and tumor-specific immunity.

Enhanced autoimmunity associated with induction of tumor immunity in thyroiditis-susceptible mice.

BACKGROUND: Immunotherapeutic modalities to bolster tumor immunity by targeting specific sites of the immune network often result in immune dysregulation with adverse autoimmune sequelae. To understand the relative risk for opportunistic autoimmune disorders, we studied established breast cancer models in mice resistant to experimental autoimmune thyroiditis (EAT). EAT is a murine model of Hashimoto's thyroiditis, an autoimmune syndrome with established MHC class II control of susceptibility. The highly prevalent Hashimoto's thyroiditis is a prominent autoimmune sequela in immunotherapy, and its relative ease of diagnosis and treatment could serve as an early indicator of immune dysfunction. Here, we examined EAT-susceptible mice as a combined model for induction of tumor immunity and EAT under the umbrella of disrupted regulatory T cell (Treg) function. METHODS: Tumor immunity was evaluated in female CBA/J mice after depleting Tregs by intravenous administration of CD25 monoclonal antibody and/or immunizing with irradiated mammary adenocarcinoma cell line A22E-j before challenge; the role of T cell subsets was determined by injecting CD4 and/or CD8 antibodies after tumor immunity induction. Tumor growth was monitored 3×/week by palpation. Subsequent EAT was induced by mouse thyroglobulin (mTg) injections (4 daily doses/week over 4 weeks). For some experiments, EAT was induced before establishing tumor immunity by injecting mTg+interleukin-1, 7 days apart. EAT was evaluated by mTg antibodies and thyroid infiltration. RESULTS: Strong resistance to tumor challenge after Treg depletion and immunization with irradiated tumor cells required participation of both CD4(+) and CD8(+) T cells. This immunity was not altered by induction of mild thyroiditis with our protocol of Treg depletion and adjuvant-free, soluble mTg injections. However, the increased incidence of mild thyroiditis can be directly related to Treg depletion needed to achieve strong tumor immunity. Moreover, when a subclinical, mild thyroiditis was induced with soluble mTg and low doses of interleukin-1, to simulate pre-existing autoimmunity in patients subjected to cancer immunotherapy, mononuclear infiltration into the thyroid was enhanced. CONCLUSIONS: Our current findings indicate that genetic predisposition to autoimmune disease could enhance autoimmunity during induction of tumor immunity in thyroiditis-susceptible mice. Thus, HLA genotyping of cancer patients should be part of any risk assessment.

Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis.

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto's thyroiditis and Graves' disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E(b) as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E(b)-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E(b) and H2E(k) molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.

Control of Th2-mediated inflammation by regulatory T cells.

Allergic diseases and asthma are caused by dysregulated Th2-type immune responses, which drive disease development in susceptible individuals. Immune tolerance to allergens prevents inflammatory symptoms in the respiratory mucosa and provides protection against inflammation in the airways. Increasing evidence indicates that Foxp3+ regulatory T cells (Tregs) play a critical role in immune tolerance and control Th2-biased responses. Tregs develop in the thymus from CD4(+) T cells (natural Tregs) and also in the periphery by the conversion of naïve CD4(+) T cells (induced Tregs). Increased susceptibility to allergy and airway inflammation is hypothesized to result from impaired development and function of Tregs. Thus, strategies to induce allergen-specific Tregs hold great promise for treatment and prevention of asthma.

Opportunistic autoimmune disorders: from immunotherapy to immune dysregulation.

Rapid advances in our understanding of the immune network have led to treatment modalities for malignancies and autoimmune diseases based on modulation of the immune response. Yet therapeutic modulation has resulted in immune dysregulation and opportunistic autoimmune sequelae, despite prescreening efforts in clinical trials. This review focuses on recent clinical data on opportunistic autoimmune disorders arising from three immunotherapeutic modalities: (1) systemic immunomodulators, including interferon-alpha (also used to treat hepatitis C patients) and interferon-beta; (2) monoclonal antibodies to CTLA-4 and CD52, and (3) hematopoietic stem cell transplantation. Uncategorized predisposing factors in these patients include major histocompatibility complex and gender genetics, prevalence of different autoimmune diseases, prior chemotherapy, underlying disorder (e.g., hepatitis C), and preconditioning regimens as part of organ and stem cell transplants. Not unexpectedly, the prevalent autoimmune thyroid disease surfaced frequently. Our combination models to study the balance between thyroid autoimmunity and tumor immunity upon regulatory T-cell perturbation are briefly described.

Autoimmune thyroiditis: a model uniquely suited to probe regulatory T cell function.

Murine experimental autoimmune thyroiditis (EAT) is a model for Hashimoto's thyroiditis that has served as a prototype of T cell-mediated autoimmunity for more than three decades. Key roles for MHC restriction and autoantigen influence on susceptibility to autoimmunity have been demonstrated in EAT. Moreover, it has served a unique role in investigations of self tolerance. In the early 1980s, self tolerance and resistance to EAT induction could be enhanced by increasing circulating levels of the autoantigen, thyroglobulin (Tg), by exogenous addition as well as endogenous release. This observation, directly linking circulating self antigen to self tolerance, led to subsequent investigations of the role of regulatory T cells (Tregs) in self tolerance. These studies revealed that protection against autoimmunity, in both naive and tolerized mice, was mediated by thymically-derived CD4(+)CD25(+)Foxp3(+) Tregs. Moreover, these naturally-existing Tregs required proper costimulation, in context with autoantigen presentation, to maintain and enhance self tolerance. In particular was the selected use of MHC- and heterologous Tg-restricted models from both conventional and transgenic mice. These models helped to elucidate the complex interplay between autoantigen presentation and MHC class II-mediated T cell selection in the development of Treg and autoreactive T cell repertoires determining susceptibility to autoimmunity. Here we describe these investigations in further detail, providing a context for how EAT has helped shape our understanding of self tolerance and autoimmunity.

Tumor regression following DNA vaccination and regulatory T cell depletion in neu transgenic mice leads to an increased risk for autoimmunity.

Modulation of the immune system to amplify anti-tumor immunity carries the risk of developing autoimmune diseases, including hypothyroidism, as seen with cancer patients undergoing clinical trials for immunotherapeutic regimens. Although there is a tendency to view autoimmunity as a positive indicator for cancer immunotherapy, some autoimmune manifestations can be life-threatening and necessitate prolonged medical intervention or removal from trial. We have established murine test models to assess such risks by monitoring, simultaneously, the immune reactivity to tumor-associated rat erbB-2 (neu) and another self Ag, mouse thyroglobulin (mTg). We previously reported that in wild-type, thyroiditis-resistant BALB/c mice that underwent regression of neu(+) TUBO tumors following regulatory T cell (Treg) depletion, immune responses to rat neu and mTg with resultant autoimmune thyroiditis (EAT) were both enhanced. In this study, we tested the balance between tumor immunity and autoimmunity in neu-transgenic BALB NeuT female mice. First, growth and progression of neu(+) tumor were compared in neu tolerant mice treated with either CD25 mAb to deplete Tregs and/or DNA vaccination. Only Treg depletion followed by neu DNA vaccination abrogated tolerance to neu, resulting in complete regression of neu(+) tumors, as well as long-term protection from spontaneous tumorigenesis in 58% of mice. The risk of developing EAT was then assessed by incorporated mTg immunization with or without LPS as adjuvant. In mice with induced tumor regression, mTg response was enhanced with modest increases in EAT development. Therefore, tumor regression induced by Treg depletion and DNA vaccination can exacerbate autoimmunity, which warrants close monitoring during immunotherapy.

Naturally-existing CD4(+)CD25(+)Foxp3(+) regulatory T cells are required for tolerance to experimental autoimmune thyroiditis induced by either exogenous or endogenous autoantigen.

Murine experimental autoimmune thyroiditis (EAT) is a model for Hashimoto's thyroiditis, an organ-specific autoimmune disease characterized by mononuclear cell infiltration and destruction of the thyroid gland. Susceptibility to EAT is MHC-linked, and influenced by CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Treg depletion enables thyroiditis induction with mouse thyroglobulin (mTg) in traditionally-resistant mice and mTg-induced, Treg-mediated tolerance protects against EAT induction in genetically-susceptible mice. Here, we demonstrate the existence of naturally-existing CD4(+)CD25(+)Foxp3(+) Tregs (nTregs) influencing thyroiditis development in naive susceptible mice and that induction of thyroiditis in these mice involves overcoming peripheral homeostatic immune suppression by nTregs. Additionally we demonstrate that nTregs are required for induction of antigen-specific tolerance, indicating that induced EAT tolerance is a result of activation of naturally-existing nTregs rather than de novo generation of induced Tregs (iTregs). Examination of several potential costimulatory molecules previously described as involved in peripheral activation of Tregs demonstrates a critical role indeed for CTLA-4 in the activation of nTregs leading to development of EAT tolerance and providing a mechanism for mTg-induced Treg activation during tolerance induction. Together, these data reinforce the important role of Tregs in mediating self-tolerance, and illuminate a potential mechanism for their therapeutic expansion in induced tolerance.

Autoimmune thyroiditis as an indicator of autoimmune sequelae during cancer immunotherapy.

Improving cancer immunotherapy by targeting T cell network also triggers autoimmunity. We disrupted regulatory T cell (Treg) function to probe the balance between breast cancer vaccination and autoimmune thyroiditis (EAT) in four models, with particular attention to MHC-associated susceptibility, EAT induction with mouse thyroglobulin (mTg) without adjuvant, and tolerance to Her-2/neu in transgenic mice. 1) In EAT-resistant BALB/c mice, Treg depletion enhanced tumor regression, and facilitated mild thyroiditis induction. 2) In Her-2 tolerant C57BL/6 mice expressing HLA-DR3, an EAT-susceptibility allele, Her-2 DNA vaccinations must follow Treg depletion for (Her-2xDR3)F(1) mice to resist tumor challenge; thyroiditis incidence was moderated by the EAT-resistant IA(b) allele. 3) In neu tolerant, EAT-resistant BALB/c mice, implanted neu(+) tumor also regressed only after Treg depletion and DNA vaccinations. Tumor immunity was long-term, providing protection from spontaneous tumorigenesis. In all three, immune stimuli from concurrent tumor regression and EAT development have a noticeable, mutually augmenting effect. 4) In Treg-depleted, EAT-susceptible CBA/J mice, strong tumor protection was established by immunization with a cell vaccine. mTg injections led to greater thyroiditis incidence and severity. Combination models with MHC class II diversity should facilitate autoimmunity risk assessment and management while generating tumor immunity.

Toward better models of hyperthyroid Graves' disease.

Graves' disease affects only humans. Although it is a treatable illness, medical therapy with antithyroid drugs is imperfect, showing high rates of recurrence. Furthermore, the etiology and treatment of the associated ophthalmopathy still represent problematic issues. Animal models could contribute to the solution of such problems by providing a better understanding of the underlying pathogenesis and could be used for evaluating novel therapeutic strategies. This article discusses the pursuit of a better experimental model for hyperthyroid Graves' disease and outlines how this research has clarified the immunology of the disease.

A novel H2A-E+ transgenic model susceptible to human but not mouse thyroglobulin-induced autoimmune thyroiditis: identification of mouse pathogenic epitopes.

The A-E+ transgenic mouse is highly susceptible to human thyroglobulin (hTg)-induced thyroiditis, but strongly tolerant to a challenge by mouse thyroglobulin (mTg), in stark contrast to traditionally susceptible strains, wherein mTg induces stronger thyroiditis. To identify mouse thyroid epitopes recognized by destructive, hTg-primed T cells, we selected the three hTg epitopes known to be presented by H2E(b), as the basis for synthesizing potential mTg epitopes. One 15-mer peptide, mTg409, did prime T cells, elicit Ab, and induce thyroiditis. Moreover, cells primed with corresponding, pathogenic hTg410 cross-reacted with mTg409, and vice versa. mTg409 contained 4/4 anchor residues, similar to the corresponding hTg peptide. Based on this finding, a second mTg epitope, mTg179, was subsequently identified. These mTg autoepitopes, identified by using thyroiditogenic hTg epitopes, help to explain the severe thyroiditis seen in this novel A-E+ transgenic model.

H2E-derived Ealpha52-68 peptide presented by H2Ab interferes with clonal deletion of autoreactive T cells in autoimmune thyroiditis.

Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2(b)) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting A(b) presentation to thyroiditogenic T cells. Yet, Ea(k) transgenic mice, expressing A(b) and normally absent E(b) molecules (E(+)B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of E(b) expression on mTg presentation by A(b), seven putative A(b)-binding, 15-16-mer peptides were synthesized. Five were immunogenic for both B10 and E(+)B10 mice. The effect of E(b) expression was tested by competition with an Ealpha52-68 peptide, because Ealpha52-68 occupies approximately 15% of A(b) molecules in E(+)B10 mice, binding with high affinity. Ealpha52-68 competitively reduced the proliferative response to mTg, mTg1677, and mTg2342 of lymph node cells primed to each Ag. Moreover, mTg1677 induced mild thyroiditis in Treg-depleted B10 mice, and in E(+)B10 mice without the need for Treg depletion. Ealpha52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.

The "A, B and C" of Her-2 DNA vaccine development.

INTRODUCTION: The development of Her-2 DNA vaccine has progressed through three phases that can be categorized as phase "A": the pursuit of Her-2 as a tumor-associated "antigen", phase "B": tilting the "balance" between tumor immunity and autoimmunity and phase "C": the on-going "clinical trials". MATERIALS AND METHODS: In phase "A", a panel of human ErbB-2 or Her-2 plasmids were constructed to encode non-transforming Her-2 derivatives. The immunogenicity and anti-tumor activity of Her-2 DNA vaccines were tested in human Her-2 transgenic mice with or without the depletion of regulatory T cells (Tregs). However, Treg depletion or other immune modulating regimens may increase the risk of autoimmunity. In phase "B", the balance between tumor immunity and autoimmunity was assessed by monitoring the development of experimental autoimmune thyroiditis (EAT). To test the efficacy of Her-2 DNA vaccines in cancer patients, clinical trials have been initiated in phase "C". RESULTS AND CONCLUSIONS: Significant anti-Her-2 and anti-tumor activity was observed when Her-2 transgenic mice were electro-vaccinated after Treg depletion. Susceptibility to EAT was also enhanced by Treg depletion and there was mutual amplification between Her-2 immunity and EAT development. Although Tregs regulate both EAT and Her-2 immunity, their effector mechanisms may differ. It may be possible to amplify tumor immunity with improved strategies that can by-pass undue autoimmunity. Critical information will be revealed in the next decade to expedite the development of cancer vaccines.

Application of HLA class II transgenic mice to study autoimmune regulation.

In the past decade, we participated in the increased use of HLA class II transgenic mice to delineate genetic control in autoimmune diseases. Our studies began with individual class II transgenes to determine permissiveness for experimental autoimmune thyroiditis (EAT), first in resistant strains and then in the absence of endogenous H2 class II molecules. Polymorphism for HLA-DRB1 was observed, as DR3, but not DR2 or DR4, molecules serve as a determinant for EAT induction with either mouse thyroglobulin (mTg) or human thyroglobulin (hTg). This delineation enabled identification of pathogenic Tg peptides, based on DR3-binding motifs. HLA-DQ polymorphism was also detectable; hTg induced moderate EAT in DQ8(+), but not DQ6(+), mice. Coexpressing permissive and nonpermissive alleles, DR3(+) mice showed reduced EAT severity in the presence of DQ8, but not DQ6, DR2, or DR4. Determining the regulatory T cell (Treg) influences showed that Treg depletion increased thyroiditis incidence and severity without altering the major histocompatibility complex-based hierarchy in susceptibility. This increase after Treg depletion can also be observed in NaI-induced thyroiditis in DR3(+) mice, a means to study a major environmental factor in thyroid autoimmunity. DR3(+) mice were also immunized with human thyroid peroxidase cDNA, resulting in thyroiditis and an antibody (Ab) profile resembling patient antibodies (Abs). Similar immunization with human TSH receptor cDNA resulted in thyroid-stimulating Abs and elevated T(4) levels with moderate thyroiditis in some animals, suggesting a potential Graves' disease model that due to thyroid lesions is more complete than other models. Recently, Treg manipulation in cancer immunotherapy trials has triggered various autoimmune disorders. Thus, DR3(+) mice are being used to monitor a known risk factor for autoimmune thyroid disease in attempts to enhance tumor immunity.

Control of Her-2 tumor immunity and thyroid autoimmunity by MHC and regulatory T cells.

Immune reactivity to self-antigens in both cancer and autoimmune diseases can be enhanced by systemic immune modulation, posing a challenge in cancer immunotherapy. To distinguish the genetic and immune regulation of tumor immunity versus autoimmunity, immune responses to human ErbB-2 (Her-2) and mouse thyroglobulin (mTg) were tested in transgenic mice expressing Her-2 that is overexpressed in several cancers, and HLA-DRB1*0301 (DR3) that is associated with susceptibility to several human autoimmune diseases, as well as experimental autoimmune thyroiditis (EAT). To induce Her-2 response, mice were electrovaccinated with pE2TM and pGM-CSF encoding the extracellular and transmembrane domains of Her-2 and the murine granulocyte macrophage colony-stimulating factor, respectively. To induce EAT, mice received mTg i.v. with or without lipopolysaccharide. Depletion of regulatory T cells (Treg) with anti-CD25 monoclonal antibody enhanced immune reactivity to Her-2 as well as mTg, showing control of both Her-2 and mTg responses by Treg. When immunized with, Her-2xDR3 and B6xDR3 mice expressing H2(b)xDR3 haplotype developed more profound mTg response and thyroid pathology than Her-2 or B6 mice that expressed the EAT-resistant H2(b) haplotype. In Her-2xDR3 mice, the response to mTg was further amplified when mice were also immunized with pE2TM and pGM-CSF. On the contrary, Her-2 reactivity was comparable whether mice expressed DR3 or not. Therefore, induction of Her-2 immunity was independent of DR3 but development of EAT was dictated by this allele, whereas Tregs control the responses to both self-antigens. These results warrant close monitoring of autoimmunity during cancer immunotherapy, particularly in patients with susceptible MHC class II alleles.

Chronic exposure in vivo to thyrotropin receptor stimulating monoclonal antibodies sustains high thyroxine levels and thyroid hyperplasia in thyroid autoimmunity-prone HLA-DRB1*0301 transgenic mice.

We have examined the induction of autoimmunity and the maintenance of sustained hyperthyroidism in autoimmunity-prone human leucocyte antigen (HLA) DR3 transgenic non-obese diabetic (NOD) mice following chronic stimulation of the thyrotropin receptor (TSHR) by monoclonal thyroid-stimulating autoantibodies (TSAbs). Animals received weekly injections over the course of 9 weeks of monoclonal antibodies (mAbs) with strong thyroid-stimulating properties. Administration of the mAbs KSAb1 (IgG2b) or KSAb2 (IgG2a), which have similar stimulating properties but different TSH-binding blocking activity, resulted in significantly elevated serum thyroxine (T(4)) levels and thyroid hyperplasia. After the first injection, an initial surge then fall in serum T(4) levels was followed by sustained elevated levels with subsequent injections for at least 63 days. Examination of KSAb1 and KSAb2 serum bioactivity showed that the accumulation of the TSAbs in serum was related to their subclass half-lives. The thyroid glands were enlarged and histological examination showed hyperplastic follicles, with minimal accompanying thyroid inflammation. Our results show that chronic in vivo administration of mAbs with strong thyroid-stimulating activity resulted in elevated T(4) levels, suggesting persistent stimulation without receptor desensitization, giving a potential explanation for the sustained hyperthyroid status in patients with Graves' disease. Moreover, despite the presence of HLA disease susceptibility alleles and the autoimmune prone NOD background genes, chronic stimulation of the thyroid gland did not lead to immune cell-mediated follicular destruction, suggesting the persistence of immunoregulatory influences to suppress autoimmunity.

Experimental autoimmune thyroiditis in the mouse.

Experimental autoimmune thyroiditis (EAT) in mice is an excellent model for Hashimoto's thyroiditis (HT). It is induced with thyroglobulin (Tg), a known thyroid autoantigen that is common to both mouse and human and for which several conserved, thyroiditogenic epitopes have been identified. This unit describes induction and evaluation of EAT using thyroid histology and in vitro proliferative response assays. An ELISA is presented to detect the level of antibody to mouse thyroglobulin (MTg). To induce EAT, either bacterial lipopolysaccharide (LPS) or supplemented complete Freund's adjuvant (CFA) can be used as adjuvant. A support protocol for preparing MTg is included. The T cell proliferation assay can be used to examine the antigenicity of synthetic peptides derived from MTg or heterologous Tg. EAT can be adoptively transferred utilizing cells that have been expanded in vitro, as described. A protocol is provided for inducing tolerance using deaggregated MTg; induction of tolerance requires larger amounts of MTg but efficiently suppresses EAT development. Also included is a protocol to demonstrate the role of regulatory T cells in mediating tolerance. A protocol to delineate HLA association with HT is illustrated using HLA class II transgenic mice.

Tolerance to autoimmune thyroiditis: (CD4+)CD25+ regulatory T cells influence susceptibility but do not supersede MHC class II restriction.

Murine experimental autoimmune thyroiditis (EAT), a model of Hashimoto's thyroiditis, has served for more than three decades as a prototypical model of T cell-mediated autoimmunity. Early investigations demonstrated a clear correlation between genetic factors, particularly the H2A locus of the MHC class II region, and susceptibility to autoimmune thyroiditis. Early studies also demonstrated that susceptibility to EAT induction could be modulated by manipulation of circulating levels of thyroglobulin (Tg), the principal thyroid antigen, resulting in the strengthening of self-tolerance. This antigen-specific induced tolerance is mediated by thymus-derived cells, and subsequent investigations revealed that the suppressive function is located in the (CD4+)CD25+ T cell subset, similar to findings in other models. We have demonstrated that these (CD4+)CD25+ regulatory T cells (Treg) influence susceptibility to thyroiditis in naive, as well as mTg-tolerized mice. Here, we describe the influence of both Treg and MHC class II haplotype, independently, as well in combination, and describe our recent utilization of MHC class II transgenic mice to directly compare the extent of their influences.

Interference with CD4+CD25+ T-cell-mediated tolerance to experimental autoimmune thyroiditis by glucocorticoid-induced tumor necrosis factor receptor monoclonal antibody.

Experimental autoimmune thyroiditis (EAT), a murine model for Hashimoto's thyroiditis, is inducible with mouse thyroglobulin (mTg), and characterized by mononuclear cell infiltration and destruction of the thyroid gland. Pretreatment with mTg leads to CD4+CD25+ T-cell-mediated resistance to subsequent EAT induction. We have recently demonstrated that in vivo administration of a monoclonal antibody (mAb) to CD137, a member of the tumor necrosis factor receptor (TNFR) family, interferes with both the development and mediation of induced EAT tolerance. Here, we examined the influence of another TNFR family member, glucocorticoid-induced TNFR (GITR), which has been reported to modulate the function of CD4+CD25+ T cells in other models. We found that in vivo administration of GITR mAb inhibited EAT tolerance induction and abrogated established tolerance, enabling thyroiditis induction. In in vitro assays, GITR mAb inhibited suppression of mTg-primed T cells by CD4+CD25+ T cells isolated from mTg-pretreated mice. The target of GITR mAb appears to be CD4+CD25- T cells, rather than CD4+CD25+ T cells from tolerized mice, suggesting that GITR signaling likely interferes with EAT tolerance by enabling thyroiditogenic T cells to circumvent suppression by CD4+CD25+ regulatory T cells.

Concurrent induction of antitumor immunity and autoimmune thyroiditis in CD4+ CD25+ regulatory T cell-depleted mice.

When CD4+ CD25+ regulatory T cells are depleted or inactivated for the purpose of enhancing antitumor immunity, the risk of autoimmune disease may be significantly elevated because these regulatory T cells control both antitumor immunity and autoimmunity. To evaluate the relative benefit and risk of modulating CD4+ CD25+ regulatory T cells, we established a new test system to measure simultaneously the immune reactivity to a tumor-associated antigen, neu, and an unrelated self-antigen, thyroglobulin. BALB/c mice were inoculated with TUBO cells expressing an activated rat neu and treated with anti-CD25 monoclonal antibody to deplete CD25+ cells. The tumors grew, then regressed, and neu-specific antibodies and IFN-gamma-secreting T cells were induced. The same mice were also exposed to mouse thyroglobulin by chronic i.v. injections. These mice produced thyroglobulin-specific antibody and IFN-gamma-secreting T cells with inflammatory infiltration in the thyroids of some mice. The immune responses to neu or thyroglobulin were greater in mice undergoing TUBO tumor rejection and thyroglobulin injection than in those experiencing either alone. To the best of our knowledge, this is the first experimental system to assess the concurrent induction and possible synergy of immune reactivity to defined tumor and self-antigens following reduction of regulatory T cells. These results illustrate the importance of monitoring immune reactivity to self-antigens during cancer immunotherapy that involves immunomodulating agents, and the pressing need for novel strategies to induce antitumor immunity while minimizing autoimmunity.