Neonatal Tolerance Induction Research Articles

Anergy-derived Foxp3+ T regulatory cell differentiation in the neonatal mouse

Abstract The neonatal period is a natural scenario to study the effects of peripheral self-antigen (Ag) recognition by autoreactive conventional CD4+ T cells under conditions of low Foxp3+ T regulatory cell (Treg) numbers and function. Moreover, the neonatal environment is an interesting scenario to study the relationship between the induction of CD4+ T cell anergy and the peripheral generation of anergy-derived Tregs, given that anergy induction relies on normal Treg function whereas anergy-derived Treg generation is optimal in the setting of Treg cell deficiency. We have hypothesized that the neonatal period is both necessary and sufficient to allow for the induction of anergy as well as the peripheral differentiation of anergy-derived Foxp3+ Tregs from naive conventional CD4+ T cells during self-Ag recognition. To test our hypotheses we developed a system of neonatal peripheral tolerance induction. Neonatal pups engineered to express the experimental “self” peptide Ag chicken ovalbumin (OVAp) were given OVAp/I-Ab–specific OT-II CD4+ T cells, and the pups were later examined for anergy induction, Treg differentiation, and development of autoimmunity. Our data demonstrate that in the neonatal period: 1) previously naive CD4+ T cells express markers of anergy following exposure to self-Ag, and a small fraction of these cells can be seen to differentiate to a Foxp3+ Treg phenotype, and 2) previously anergic CD4+ T cells typically retain their anergic phenotype but also give rise to a substantial fraction of Foxp3+ Tregs, and 3) animals remain free of autoimmune disease. These findings suggest that during the neonatal period peripheral Foxp3+ Treg cell differentiation may proceed through a conventional anergic CD4+ T cell intermediate.

Immune impact of commensals versus pathobionts

Pathobiont but not commensal cutaneous colonization elicits an inflammatory response that prevents neonatal induction of microbial tolerance in the skin. Pathobiont but not commensal cutaneous colonization elicits an inflammatory response that prevents neonatal induction of microbial tolerance in the skin. CITATION Leech JM, Dhariwala MO, Lowe MM, et al. Toxin-triggered interleukin-1 receptor signaling enables early-life discrimination of pathogenic versus commensal skin bacteria. Cell Host Microbe. 2019;26:795–809. How the body maintains tolerance to commensals but not to microbes with pathogenic potential (pathobionts) such that appropriate immune responses can be mounted to the pathogens in case of barrier breach without triggering chronic inflammation to the commensals remains to be determined. Leech et al. demonstrate that neonatal skin colonization with the commensal Staphylococcus epidermidis (S. epi) induces a greater regulatory T cell (Treg) response than colonization with the pathobiont Staphylococcus aureus. The higher effector-to-Treg ratio triggered by S. aureus is due to S. aureus’ virulence factor α-toxin–dependent induction of the inflammatory cytokine IL-1β. Tregs are found in high abundance at barrier sites. The authors had previously found a temporal window after birth in which cutaneous S. epi colonization of mouse pups could elicit Treg recruitment to skin hair follicles, an event necessary to establish tolerance to S. epi and prevent immune responses to S. epi rechallenge during skin abrasion in adulthood. To compare immune responses to S. epi and S. aureus, the authors engineered the bacteria to express the model antigen 2W fused to fluorescent mCherry protein and tracked 2W-specific T cell responses with 2W:I-Ab tetramers by flow cytometry. mCherry fluorescence demonstrated similar bacterial growth and persistence of the two strains. Neonatal skin colonization with S. epi but not S. aureus resulted in protection against inflammation and neutrophil accumulation following adult bacterial challenge and skin abrasion, and a reduced frequency of Tregs among 2W-reactive CD4+ T cells was observed in the skin-draining lymph node (sdLN) of S. aureus- versus S. epi-colonized mice. Differences were not due to distinct types of skin dendritic cells picking up S. aureus versus S. epi. To investigate whether a different quality in immune priming to S. aureus than S. epi was responsible for the lack of tolerance elicited by S. aureus, the authors performed RNAseq in bulk skin Tregs and T effectors of colonized mice. Both T effectors and Tregs following S. aureus colonization were enriched in genes associated with type 1 interleukin (IL) receptor signature, and elevated levels of IL-1β transcripts were found in S. aureus-colonized skin. Supporting the importance of this pathway, 2W-reactive Treg percentages and numbers were increased in the sdLN of S. aureus-colonized Il1r-/- mice. Conversely, cutaneous application of IL-1β at the time of S. epi colonization reduced the percentage of Tregs this colonization elicited, although whether this prevented induction of tolerance to S. epi was not reported. To explore potential S. aureus virulence factors responsible for inducing active IL-1β, the authors employed S. aureus strains lacking virulence factors. Lack of α-toxin correlated with reduced production of IL-1β by myeloid cells in vitro and increased Treg induction by S. aureus colonization, whereas α-toxin application reduced Treg induction by S. epi, although the impact on tolerance was not tested. Overall, virulence factors encoded by S. aureus may be responsible for activation of the inflammasome and consequent production of active IL-1β may limit Treg induction by S. aureus colonization, thus ensuring more robust immune responses to S. aureus during subsequent barrier breach. This may be one way by which the immune system distinguishes commensals from pathobionts. Colonized organs, such as lung and intestine, have a shorter half-life following transplantation than sterile organs, such as kidney and heart, suggesting the hypothesis that microbial colonization may influence graft damage. How the recipient of a colonized graft discriminates between host commensals, donor commensals brought in by the graft and pathogens that may colonize the donor organ or the recipient’s, depending on the underlying disease driving transplantation, remains to be determined. Whether donor Tregs present in the donor organ can protect the graft recipient from excessive inflammatory immune responses to donor commensals after transplantation might be important to understand. These types of studies may help explain correlations between the presence of specific microbial taxa and the progressive loss of graft function. Dr. Alegre is a professor in the Department of Medicine at the University of Chicago. She is also section editor of “Literature Watch.”

Influences of Maternal Factors Over Offspring Allergies and the Application for Food Allergy.

The prevalence of food allergy has been steadily rising worldwide with the highest incidence noted among younger children, and increasingly recognized as a growing public concern. The first known ingestion of foods often causes allergic reaction, suggesting that sensitization of offspring with food allergens may occur during pregnancy and/or through breastfeeding. This creates a milieu that shapes the neonatal immune responses to these allergens. However, the effects of maternal allergen exposure and maternal sensitization with allergens on development of allergies in offspring remain controversial. This review discusses recent advances from human data in our understanding of how maternal factors, namely, food allergens, allergen-specific immunoglobulins, cytokines, genetics, and environmental factors transferred during pregnancy or breastfeeding influence offspring allergies and how such effects may be applicable to food allergy. Based on information obtained from mouse models of asthma and food allergy, the review also dissects the mechanisms by which maternal factors, including the impact of immune complexes, transforming growth factor-β, vitamin A, and regulatory T-cell responses, contribute to the induction of neonatal tolerance vs. development of allergic responses to maternally transferred allergens.

Induction of neonatal tolerance to GFP-labeled karyocytes in C57/B6 mice

Green fluorescent protein is widely used in biological studies including parabiosis models for visualization of cellular structures and cells. However, the growing number of the data is available regarding immunogenicity of this protein, which can interfere with its use in in vivo experiments. In this study, we attempted to induce neonatal immunological tolerance to GFP-labeled karyocytes by intraperitoneal injections of B6.GFP mouse splenocytes to newborn C57/B6 mice. GFP+ skin graft integrity was evaluated under UV light at 6weeks after skin grafting. GFP+ skin transplants survived up to 6weeks after grafting in all animals that undergone neonatal tolerance induction, whereas all skin grafts were rejected in control naïve mice within first two weeks. Thus, current protocol is suitable for induction of immune tolerance against GFP-labeled karyocytes.

Neonatal tolerance induction enables accurate evaluation of gene therapy for MPS I in a canine model.

High fidelity animal models of human disease are essential for preclinical evaluation of novel gene and protein therapeutics. However, these studies can be complicated by exaggerated immune responses against the human transgene. Here we demonstrate that dogs with a genetic deficiency of the enzyme α-l-iduronidase (IDUA), a model of the lysosomal storage disease mucopolysaccharidosis type I (MPS I), can be rendered immunologically tolerant to human IDUA through neonatal exposure to the enzyme. Using MPS I dogs tolerized to human IDUA as neonates, we evaluated intrathecal delivery of an adeno-associated virus serotype 9 vector expressing human IDUA as a therapy for the central nervous system manifestations of MPS I. These studies established the efficacy of the human vector in the canine model, and allowed for estimation of the minimum effective dose, providing key information for the design of first-in-human trials. This approach can facilitate evaluation of human therapeutics in relevant animal models, and may also have clinical applications for the prevention of immune responses to gene and protein replacement therapies.

Neonatal Immune Tolerance Induction to Allow Long-Term Studies With an Immunogenic Therapeutic Monoclonal Antibody in Mice.

The purpose of this study is to test the feasibility of neonatal immune tolerance induction in mice to enable long-term pharmacokinetic studies with immunogenic therapeutic monoclonal antibodies (mAb). Neonatal immune tolerance was induced by transfer of a mAb to neonatal mice via colostrum from nursing mother mice treated with two subcutaneous doses of a tolerogen starting within the first 24 h after delivery. Adalimumab and efalizumab were administered as tolerogens at various dose levels. Tolerance induction was evaluated in the offspring after reaching adulthood at 8 weeks of age. After a single intravenous injection of the same mAb as used for tolerance induction, the pharmacokinetics of the mAb and formation of anti-drug antibodies (ADA) in plasma were assessed using ELISA. Tolerance induction to adalimumab was achieved in a maternal dose-dependent manner. Adalimumab immune-tolerant offspring showed a slower adalimumab clearance (4.24 ± 0.32 mL/day/kg) as compared to the control group (12.09 ± 3.81 mL/day/kg). In the control group, accelerated clearance started 7 days after adalimumab dosing, whereas immune-tolerant offspring showed a log-linear terminal concentration-time course. In the offspring, the absence of predose ADA levels was indicative of successful tolerance induction. The second test compound efalizumab was not immunogenic in mice under our experimental conditions. Overall, the present study demonstrated the suitability of neonatal immune tolerance induction for a 4-week single dose study in adult mice with a human therapeutic mAb that is otherwise immunogenic in laboratory animals.

Failure of Neonatal B-Cell Tolerance Induction by ABO-Incompatible Kidney Grafts in Piglets

ABO-incompatible (ABOi) infant heart transplantation results in B-cell tolerance to graft A/B antigens, confirming human susceptibility to acquired immunologic or "neonatal" tolerance as described originally in murine models. Starting with this clinical observation, we sought to model neonatal ABOi organ transplantation to allow mechanistic studies of tolerance. Plasma anti-A/B antibodies were measured over time in piglets to establish developmental antibody kinetics. Blood group O piglets received kidney allografts from group A (AO-incompatible) or group O (AO-compatible) donors under cyclosporine immunosuppression. Anti-A antibodies were measured serially after transplantation; A/H antigen expression and allograft rejection were assessed in graft biopsies. Anti-A antibodies developed in naïve piglets in a kinetic pattern analogous to human infants; anti-B remained low. After transplantation, anti-A antibodies developed similarly in AO-incompatible and AO-compatible groups and were not suppressed by cyclosporine. A/H antigen expression was persistent in all graft biopsies; however, A/H antigens were not detected in vascular endothelium. Cellular and antibody-mediated rejection was absent or minimal in early and late biopsies in both groups, with one exception. Naturally delayed isohemagglutinin production in piglets is analogous to the developmental kinetics in human infants. However, in contrast to deficient anti-A antibody production as seen long-term after "A-into-O" infant heart transplant recipients, normal anti-A antibody production after "A-into-O" piglet kidney transplantation indicates that tolerance did not develop despite graft A antigen persistence. These findings suggest that the impact on the host immune system of exposure to nonself ABH antigens during early life in human heart versus porcine kidney grafts may depend on expression in vascular endothelium.

289 In Situ Imaging of Neonatal Tolerance Induction: Donor Bone Marrow and Spleen Cells Home Differently and Induce Tolerance by Distinct/Overlapping Mechanisms

289 In Situ Imaging of Neonatal Tolerance Induction: Donor Bone Marrow and Spleen Cells Home Differently and Induce Tolerance by Distinct/Overlapping Mechanisms

The potential role of preventing atherosclerosis by induction of neonatal tolerance to VLDL

Induction of immune tolerance to ox-LDL could reduce atherosclerosis by modulation immune response. We suppose that very low density lipoprotein (VLDL) may have a similar role to ox-LDL in autoimmune response of atherosclerosis. In this study, neonatal rats were injected with ox-LDL, VLDL, or equal-volume saline, respectively. Vaccination with ox-LDL reduced the level of specific antibody, T cells proliferation response, and the level of endothelins. The method also had a tendency of reducing blood lipids. Vaccination with VLDL obviously reduced the level of specific antibody and T cells proliferation. Though there was also a tendency of reducing blood lipids and endothelins, the effect was less prominent than that with ox-LDL. We conclude that, although the effect was less obvious, vaccination with VLDL to induce neonatal tolerance had an effect on modulating immune response, protecting endothelial cells, and reducing blood lipids.

Mechanisms of tolerance

Mechanisms of tolerance

300 Active Roles of F1 Tolerizing Cells in Early In Vivo Events Leading to Neonatal Tolerance Induction

300 Active Roles of F1 Tolerizing Cells in Early In Vivo Events Leading to Neonatal Tolerance Induction

Induction of Murine Neonatal Tolerance Against Graves' Disease Using Recombinant Adenovirus Expressing the TSH Receptor A-Subunit

Graves' disease is a common organ-specific autoimmune disease. The identity of its autoantigen, the TSH receptor (TSHR), was established and used to induce a typical animal model. A-subunit, the shed portion of TSHR, either initiates or amplifies the autoimmune response of the thyroid gland, thereby causing Graves' disease in humans. In the present study, we investigate the effect of the TSHR A-subunit on the induction of murine neonatal tolerance for the development of Graves' disease. Female BALB/c mice were pretreated with different doses of adenovirus expressing the A-subunit of TSHR (Ad-TSHR289) by either ip or im injection within the first 24 h after their birth. Graves' disease was induced after the animals reached adulthood. Nearly all mice pretreated with the high dose of Ad-TSHR289 failed to develop TSHR antibodies, detected by the TSH-binding inhibition assay, hyperthyroidism, and thyroid follicular hyperplasia. The mice preimmunized im with the lower doses of Ad-TSHR289 developed a relatively low level of TSH-binding inhibition and the low incidence of hyperthyroidism. Accordingly, the percentages of splenic CD4+CD25+/CD4+ and CD25+Foxp3+/CD4+ Treg cells were increased in mice pretreated with the high dose of Ad-TSHR289. Taken together, our data strongly indicate that the immunotolerance against Graves' disease could be induced in neonatal mice using a specific TSHR antigen in a high dose either by ip or im injection, preventing the development of Graves' disease.

B Cells Are Dispensable for Neonatal Transplant Tolerance Induction

Prior studies have demonstrated that neonatal B cells possess unique immunoregulatory properties. Specifically, neonatal B-1 cells produce interleukin (IL)-10 in response to toll-like receptor stimulation; this modulates innate and adaptive alloimmune responses. Here, we examine whether this process plays a critical role in neonatal transplant tolerance induction. We used a murine model of neonatal transplant tolerance induction, whereby female C57BL/6 mice injected with male spleen cells 3 days after birth acquire the ability to accept a male skin transplant in adulthood. We investigated the role of B cells in this model by using mice with targeted genetic B-cell deficiencies (including muMT, JH-/-, and XID mice). In addition, we examined the role of IL-10 in this model using IL-10-/- mice. Transplant tolerance induction was neither dependent on B cells nor on IL-10. Despite the role of neonatal B cells in suppressing innate and adaptive alloimmune responses, B cells are dispensable for neonatal transplant tolerance induction in this experimental model.

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: analysis of the role of donor T cells in the induction of autoimmunity

The injection of (C57BL/6 x BALB/c) F1 spleen cells into BALB/c newborn mice leads to activation of persisting F1 donor B cells and development of a lupus-like syndrome in tolerized BALB/c mice. This syndrome is characterized by hypergammaglobulinaemia, high levels of anti-DNA and anti-Sm antibodies, circulating immune complexes and deposits of immunoglobulin in renal glomeruli. The role of donor T cells in this model was investigated by injecting the newborn mice with F1 cells depleted in different T cell subsets by using specific monoclonal antibodies (MoAbs). Tolerance, as shown by an absence of H-2b-specific CTL alloreactivity and persistence of immunoglobulin bearing the donor allotype were observed in mice injected with F1 cells previously depleted in the CD4+ and/or CD8+ T cell subsets as well as in those which received Thy-1+-depleted F1 spleen cells. In these mice, a typical autoimmune syndrome was found, including splenomegaly and lymphadenopathy, anti-ssDNA and anti-aortic myosin IgG antibodies and renal deposition of immunoglobulin. However, some quantitative changes were seen: the levels of anti-aortic myosin antibodies were lower in mice tolerized with CD4+-depleted F1 cells than in those receiving untreated F1 cells. Conversely, higher levels of these autoantibodies were observed in mice tolerized with CD8+-depleted F1 cells. These results suggest that mature donor T cells are not necessary neither for the establishment of neonatal tolerance to alloantigens nor for the activation of F1 donor B cells in the production of the autoimmune syndrome in tolerant mice, but they may contribute in the regulation of the expression of autoreactive B cell clones.

327: Effect of MHC class II expression on chimerism in allogeneic fetal liver treated mice

Neonatal injection of allogeneic fetal liver cells (FLC) or semi-allogeneic bone marrow/spleen cells (F1BMSC) induces permanent acceptance of donor-type and third-party cardiac allografts. However persistent microchimerism could only be demonstrated after F1BMSC treatment, which also induces donor-type only skin graft survival, whereas FLC do not. This study examined the effect of recipient MHC expression on differential development of chimerism after neonatal tolerance induction.

Induction of xenogeneic neonatal tolerance to transgenic human leukocyte antigen class I grafts.

The immune response against xenografts is vigorous and poorly controlled with conventional immunosuppressants. Therefore, success in xenotransplantation will depend on developing additional approaches such as induction of immunologic unresponsiveness or tolerance. Although classic protocols of neonatal tolerance induction in mice are very tolerogenic in many allogeneic models, they have generally failed in xenogeneic models. The purpose of these studies was to determine whether failure results from an intrinsic property of xenogenic major histocompatibility complex (MHC) molecules themselves or, instead, is caused by some limitation in species-specific molecular interactions distinct from the polymorphic domains of xenogenic MHC molecules. Our approach was to test the ability of lymphoid cells from a transgenic (Tg) mouse donor expressing a xeno-MHC class I molecule encoding the polymorphic alpha1/alpha2 for human leukocyte antigen (HLA)-B7 to induce neonatal tolerance in non-Tg syngeneic C57BL/6 recipients. Because the donor and recipient strains are genetically identical (C57BL/6, H-2b) except for Tg human MHC HLA-B7, any species-specific molecular incompatibility in this mouse anti-human class I xeno-combination that could potentially interfere with induction of tolerance has been eliminated. Our results show that HLA-B7 Tg-, but not C57BL/6 syngeneic-, injected neonates were unresponsive as adults to HLA-B7-expressing target cells in vitro and specifically accepted HLA-B7-expressing Tg skin grafts. In addition, neonatal injection of donor cells resulted in peripheral chimerism. These experiments demonstrate that, as long as species-specific molecular interactions are maintained, recognition of the polymorphic domains of xenogeneic MHC does not represent a barrier to neonatal tolerance induction.

The ratio of n-6 to n-3 fatty acids in maternal diet influences the induction of neonatal immunological tolerance to ovalbumin.

Prevalence of allergy is increasing in many countries and might be related to changed environmental factors, such as dietary fatty acids (FA). The present study investigates whether dietary ratio of n-6 to n-3 FA influences the induction of immunological tolerance to ovalbumin (OA) in neonatal rats. During late gestation and throughout lactation Sprague-Dawley rats were fed a diet containing 7% linseed oil (n-3 diet), sunflower oil (n-6 diet) or soybean oil (n-6/n-3 diet). At 10-16 days of age the rat offspring were subsequently exposed, or not, to OA via the milk. The offspring were weaned onto the same diets as the mothers and immunized with OA and the bystander antigen human serum albumin (HSA). In the offspring on the n-3 diet exposure to OA via the milk resulted in lower delayed type hypersensitivity reaction (DTH) and antibody responses against both OA and HSA, compared to those in the offspring not exposed to OA, indicating the induction of oral tolerance. In the offspring on the n-6 diet, the exposure to OA led to depressed specific immune responses against only OA, not HSA. In the offspring on the n-6/n-3 diet oral exposure to OA did not influence immune responses against OA, or HSA. The results indicate that the dietary ratio of n-6/n-3 FA is important for the induction of neonatal oral tolerance. Thus nonoptimal feeding may have effects on the development of immunological tolerance to dietary antigen ingested by the mother. The ratio of n-6/n-3 FA in the diet may be considered in the context of increased prevalence of allergy.

1045. Neonatal Tolerance Induction by Repetitive DNA Vaccination

Background and Aims: Host immune response against the transgenic protein plays a crucial role in the success of gene therapy of inherited diseases. In addition to use immunosuppressant therapy or immunomodulation, we try to induce neonatal tolerance by repetitive DNA vaccination to attenuate host immune responses.

Defects in the regulation of B cell apoptosis are required for the production of citrullinated peptide autoantibodies in mice.

Protein deimination, a process to modify arginine residues to citrulline by the addition of a neutral oxygen group, is associated with apoptosis. The presence of autoantibodies recognizing citrullinated peptides is highly specific to rheumatoid arthritis (RA) and is therefore a useful marker for the early diagnosis of RA. In this study, we explored whether anti-cyclic citrullinated peptide (anti-CCP) autoantibodies are produced in several experimental models of autoimmune diseases in mice. The levels of anti-CCP autoantibodies were analyzed by enzyme-linked immunosorbent assay in several lupus-prone strains of mice, in animals with type II collagen (CII)-induced arthritis, and after induction of neonatal tolerance to alloantigens. We observed the production of these autoantibodies in 2 different lupus-prone mice, MRL-lpr/lpr and (NZW x B6)F(1)-hbcl-2 transgenic mice, characterized by the presence of abnormalities in the regulation of B cell apoptosis. Other genetic defects, determining autoimmune susceptibility, present in MRL and NZW mice were additionally required for anti-CCP autoantibody production. The induction of autoantibodies in normal BALB/c mice injected at birth with semiallogeneic spleen cells from (BALB/c x B6)F(1)-hbcl-2 transgenic mice suggested that these additional autoimmune defects may be related, at least in part, to the establishment of abnormal interactions between T cells and B cells. In addition, anti-CCP autoantibodies were not produced in the course of CII-induced arthritis, an experimental model of RA in mice. Our study provides evidence for the association between defects in the regulatory cell death machinery of B lymphocytes and the production of certain autoantibody specificities.