Key Points
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Monogenic defects have been identified that interfere with self tolerance.
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Human defects of self tolerance can affect both central and peripheral tolerance.
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Linking specific genetic defects to resulting autoimmunity patterns reveals specific self tolerance pathways.
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Additional genetic/environmental factors appear to impact monogenic autoimmune processes.
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It is likely that additional genetic defects resulting in autoimmunity will be identified.
Down-regulation of the immune response has become a subject of increased focus with this area of investigation complementing extensive work done to characterize the differentiation and activation of immune cells. Recently described prototypic human disorders that affect various immunoregulatory pathways have provided important insights into mechanisms required for tolerance and the control of immune responses. In this chapter we will discuss congenital disorders that impact central deletion of autoreactive T cells in the thymus as well as those that impact other mechanisms involved in the maintenance of tolerance in the periphery. In all of these disorders the clinical phenotype includes autoimmunity together with other manifestations.
Autoimmune Polyendocrinopathy, Candidiasis, Ectodermal Dystrophy (APECED)
APECED (OMIM #240300) is the prototypic disorder of defective central immune tolerance. It is an autosomal recessive disorder characterized by systemic autoimmunity that primarily affects endocrine organs, particularly the parathyroid and adrenal glands. Hypoparathyroidism, adrenal insufficiency and chronic mucocutaneous candidiasis typically characterize the syndrome but patients may also have type 1 diabetes, gonadal failure, pernicious anemia, autoimmune hepatitis and cutaneous manifestations ( Box 13-1 ).
Immunopathogenesis of APECED Syndrome
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Endocrinopathy: hypocalcemia and adrenal insufficiency caused by autoimmunity to parathyroids and adrenals
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Chronic mucocutaneous candidiasis (CMC): caused by neutralizing autoantibodies to IL-17, IL-22 and other cytokines
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Autoimmunity: thymic selection defect prevents appropriate elimination of autoreactive T cells and generation of regulatory T cells
Genetics and Immunopathogenesis
APECED is caused by mutations in the gene encoding the autoimmune regulator (AIRE), a transcription factor that plays a role in ectopic expression of tissue-specific antigens by thymic medullary epithelial cells (mTEC). In mice, AIRE-mediated self-antigen expression in the thymus has been shown to play a significant role in negative selection of autoreactive T cell clones. The mechanism by which AIRE causes expression of tissue-specific gene products may be by regulating large-scale access to chromatin.
Because naturally arising T regulatory (T REG ) cells are also thymically derived, AIRE may also play a role in generation of T REG cells. This is supported by a transgenic mouse model with a monospecific T cell receptor: autoimmunity seen in Aire −/− mice results from a combination of defective negative selection and defective generation of antigen-specific T REG cells. The role of AIRE in generation and function of T REG cells has also been investigated in APECED where a decreased percentage of CD4 + CD25 high T cells was found. In addition, CD4 + CD25 high T cells expressed less FOXP3 than control cells. Furthermore, isolated T REG cells from APECED patients had a decreased ability to suppress proliferation of effector T cells in vitro. These data suggest that AIRE plays a significant role in the generation of functional T REG cells in humans.
In addition to the recognized effector and regulatory T cell abnormalities observed in APECED, patients have a propensity to develop a broad range of pathogenic, tissue-specific autoantibodies including those directed at the parathyroids, adrenals, ovaries, lungs, gut and others. In addition, neutralizing autoantibodies against type I interferons (α and ω), interleukin-17 (IL-17), and interleukin-22 (IL-22) have been identified in many APECED patients and are associated with chronic mucocutaneous candidiasis.
Diagnosis and Treatment
APECED is typically suspected in patients who have two of the three basic symptoms: hypoparathyroidism (usually manifested by hypocalcemia), adrenal insufficiency and mucocutaneous candidiasis. Suspicion is raised further by the presence of other autoimmune manifestations and a definitive diagnosis can be made by sequencing of the AIRE gene.
Despite the impressive autoimmune phenotype, most of the therapy for APECED has focused on symptomatic treatment including calcium supplementation, steroid replacement and the management of diabetes and other endocrinopathies. Particularly problematic is the mucocutaneous candidiasis, which causes significant morbidity and increases the risk of oral malignancies. In many patients, the candidal species develop reduced sensitivity to azole antifungals over time.
Immunosuppressants are not routinely used in APECED unless patients develop autoimmune hepatitis or renal disease, in which azathioprine and cyclosporin A (cyclosporine) have shown benefit. Recent studies in Aire −/− mice have demonstrated a significant role for B cells and autoantibodies in pathogenesis, and one recent case report demonstrated efficacy of B cell depletion therapy in an APECED patient with autoimmune lung disease.
Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked (IPEX)
IPEX syndrome (OMIM #304930) is the prototype of defective peripheral immune tolerance. The basic clinical triad of IPEX includes autoimmune enteropathy, early onset endocrinopathy and dermatitis ( Box 13-2 ). The enteropathy typically presents early in life as watery diarrhea, frequently resulting in malnutrition and failure to thrive. Type 1 diabetes is the most common endocrinopathy but clinical and/or laboratory evidence of thyroiditis is also common. Eczema is the most common dermatitis in IPEX but erythroderma, psoriasiform dermatitis and pemphigus nodularis have also been observed.
In addition to the ‘IPEX triad’, most patients with IPEX also have other associated autoimmune disorders including autoimmune cytopenias, nephropathy or hepatic disease (Torgerson & Ochs, unpublished data). These conditions contribute substantially to the morbidity of patients with IPEX and increase the risk of death from disease. Patients with the classical form of the disease typically die secondary to malnutrition, electrolyte imbalance or infection before the age of 2 if not treated with aggressive immunosuppression.
Genetics and Immunopathogenesis
IPEX is caused by mutations in the forkhead DNA-binding protein FOXP3, which is expressed by CD4 + CD25 high regulatory T cells and is required for T REG cells to develop suppressor function. This has been shown most elegantly in two separate knock-in mouse models with CD4 + T cells unable to express functional Foxp3. Despite this, the cells still acquired the expected cell surface phenotype of a T REG (CD25 high CTLA-4 high GITR high ) but had no suppressive function and developed a gene expression profile suggestive of an effector/cytotoxic T cell resulting in systemic autoimmunity similar to Foxp3 −/− mice.
Under quiescent conditions, FOXP3 expression is restricted primarily to T REG cells, however it can also be inducibly expressed in a large percentage of human T cells upon activation. Originally shown to be a transcriptional repressor acting on key cytokine genes, recent genome-wide screening approaches suggest that FOXP3 functions more commonly as a transcriptional enhancer.
Most pathologic mutations in FOXP3 cluster in three important functional domains of the protein: the C-terminal forkhead DNA-binding domain, the leucine zipper and the N-terminal repression domain. Recent studies suggest that FOXP3 physically and functionally interacts with other transcription factors including NFAT, NF-κB, AML-1/RUNX1 and the retinoic acid receptor related orphan receptors RORα and RORγt to modulate gene transcription at key cytokine promoters.
Diagnosis and Treatment
IPEX is generally suspected in any patient who demonstrates at least two of the three basic clinical features of IPEX including enteropathy, endocrinopathy (type 1 diabetes mellitus or thyroiditis) and dermatitis. Flow cytometry using intracellular staining for FOXP3 protein to identify FOXP3 + T REG cells is a valuable tool to rapidly screen for the absence of T REG . Approximately 5–7% of the CD4 + T cell population is positive for FOXP3 expression in healthy controls and a marked decrease suggests a diagnosis of IPEX that may be confirmed by sequencing of the FOXP3 gene ( Figure 13-1 ). Definitive diagnosis involves identification of a mutation in FOXP3 (Torgerson, unpublished data).
From a clinical laboratory standpoint, the most consistent abnormality among IPEX patients is markedly elevated IgE while IgA is also modestly elevated in more than 50% of patients (Torgerson, unpublished data). There are no consistent abnormalities in absolute lymphocyte numbers and T cells usually proliferate normally in vitro.
Adoptive transfer studies in mice have demonstrated that the CD4 + T cells from an affected Foxp3 −/− male are capable of recapitulating the disease phenotype in a lymphopenic recipient. Treatment of IPEX has therefore focused primarily on suppression of unregulated, auto-aggressive T cells using cyclosporin A, tacrolimus (FK506) or sirolimus (rapamycin). These are often combined with steroids and/or other immunomodulatory agents including methotrexate or azathioprine. In cases where there is evidence for pathogenic autoantibodies, rituximab (anti-CD20) has proven effective. Immunosuppression is often effective initially and there is one report of a patient with IPEX being maintained for a prolonged period; however, most patients ultimately fail therapy. Currently, bone marrow transplantation holds the only hope for a long-term cure and reduced intensity regimens seem to be associated with better survival. Rapid diagnosis and transplantation early in the course of disease, before the pancreatic islet cells are destroyed, should be the goal.
STAT1 Gain of Function (STAT1-GOF) Mutations
The signal transducer and activator of transcription (STAT) family of DNA-binding proteins are critical mediators of cytokine and growth factor signaling in cells. STAT1 is the primary transcription factor activated by interferons so it plays a major role in normal immune responses, particularly to viral, mycobacterial and fungal pathogens. Recently described autosomal dominant mutations that lead to a gain of STAT1 function (STAT1-GOF; OMIM #614162) typically present with CMC but may also have disseminated infections with dimorphic yeast or mycobacteria and variable forms of autoimmunity. Recently, STAT1-GOF mutations were identified in a cohort of patients with IPEX-like autoimmunity (severe early-onset enteropathy, dermatitis, thyroiditis and type 1 diabetes). All but one of the identified patients also had a history of CMC, although in some cases the CMC was mild or only occurred after treatment with antibiotics.
In addition to IPEX-like autoimmunity and CMC, patients had recurrent respiratory infections and bronchiectasis, herpesviral infections (HSV and VZV), short stature with growth-hormone insufficiency, and arterial aneurysms (cardiac and CNS) ( Box 13-3 ). Unlike IPEX, patients with STAT1-GOF mutations had more evidence of humoral immune dysfunction including poor vaccine responses, with more than half of the patients requiring treatment with immunoglobulin replacement therapy, and normal (or mildly elevated) serum IgE levels.
Immunopathogenesis of STAT1 Gain-of-Function Syndrome
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Chronic mucocutaneous candidiasis (CMC): abnormal generation of functional Th17 cells
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Immunodeficiency: some patients make insufficient antibody responses to vaccination
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Autoimmunity: IPEX-like enteropathy and endocrinopathies, may be related to hyperactivation of STAT1 in response to interferons
Genetics and Immunopathogenesis
Inheritance is autosomal dominant, caused by heterozygous missense mutations in the STAT1 gene. All of the identified mutations that cause a gain of STAT1 function lie within the coiled-coil or DNA-binding domains of the STAT1 protein and many are associated with increased phosphorylation and delayed dephosphorylation of STAT1. In most patients, the percentage of IL-17 secreting Th17 cells in the CD4 + T cell population is markedly reduced but not absent, likely contributing to the susceptibility to CMC. The mechanism of susceptibility to autoimmunity in this disorder is, however, less clear. Regulatory T cells are present in normal to near-normal numbers and appear to have normal function based on a limited number of evaluated patients.
Diagnosis and Treatment
Hyperphosphorylation and delayed dephosphorylation of the critical tyrosine residue at position 701 (pY701) has been observed in cytokine-stimulated cells from many patients with STAT1-GOF mutations and can be measured by flow cytometry. Unfortunately, the utility of this assay as a screening test to identify patients with STAT1-GOF mutations is not yet clear, as it is not known whether the phosphorylation defect is consistent. As a result, sequencing of the STAT1 gene remains the gold standard for diagnosis.
Treatment of CMC in this disorder typically requires chronic or intermittent antifungal therapy, with mixed success. Treatment of the IPEX-like autoimmunity requires aggressive immunosuppression although there has not been a regimen or class of agents that has shown consistent efficacy. Steroids, B cell depletion therapy, and T cell directed immunosuppression (tacrolimus, cyclosporine, etc.) have all been utilized with varying results. Bone marrow transplantation (BMT) using a matched sibling donor and a reduced intensity conditioning regimen has been reported in one Peruvian patient, with transient clinical improvement, but the patient ultimately died of a presumed respiratory infection following graft rejection. At least three other patients with STAT1-GOF mutations have undergone successful BMT but are not yet reported suggesting BMT may be a viable approach in patients with severe disease.
Defects in IL-2 Signaling
Since the realization that mice lacking CD25 (the α-chain of the IL-2 receptor) have a phenotype similar to Foxp3 −/− mice, there has been a suspicion that defects that blunt IL-2 signaling in T cells might lead to an IPEX-like presentation in humans ( Figure 13-2 ).
CD25 Deficiency
Three unrelated patients with CD25 deficiency (OMIM #606367) have now been described. Similar to IPEX, all three patients developed severe, chronic diarrhea and villous atrophy in infancy (1 month, 6 weeks and 8 months of age). Two of the patients developed endocrinopathies including early-onset type 1 diabetes and thyroiditis and two developed significant skin disease including eczema, pemphigus nodularis and psoriasiform dermatitis. All three patients developed autoantibodies, hepatosplenomegaly, lymphadenopathy and lymphocytic infiltrates in various organs indicative of ongoing immune dysregulation. Unlike IPEX patients, serum IgE levels were either mildly elevated or normal.
In addition to autoimmune features, all three CD25-deficient patients had infectious complications suggestive of a more extensive defect in cellular immunity ( Box 13-4 ). Early-onset, recurrent CMV infections occurred in all patients although persistent thrush, candidal esophagitis, chronic gastroenteritis, Pseudomonas , staphylococcal and EBV infections were also seen. One patient even failed to reject an allogeneic skin graft.
Genetics and Immunopathogenesis
Inheritance is autosomal recessive leading to a complete lack of CD25 protein expression on activated T cells. Mutations included a homozygous four base pair deletion in the coding region of CD25, a homozygous mutation resulting in a single amino-acid substitution (p.S166N) and a compound heterozygous mutation.
Recent studies in Cd25 −/− mice have demonstrated that T REG cell development is normal. These cells have normal suppressive function in vitro but survival, maintenance and competitive fitness of the mature T REG cells is abnormal resulting in immune dysregulation. Future efforts to assess T REG cells in CD25-deficient patients should help to determine whether a similar mechanism is at play in humans.
Diagnosis and Treatment
All patients described to date lacked CD25 expression on T cells, suggesting that flow cytometry is an effective screening tool. Sequencing of the CD25 gene is, however, recommended to confirm the diagnosis.
Because of the ‘SCID-like’ features of this syndrome, one patient underwent a successful bone marrow transplant from a matched sibling donor and has done well. It is possible, however, that patients may respond to IL-2 therapy via the remaining low-affinity IL-2 receptor since the T cell proliferative defect was corrected in one patient ex vivo with high dose IL-2 or IL-15.
STAT5B Deficiency
Deficiency of the STAT5B transcription factor (OMIM #245590) causes a rare autosomal recessive disorder reported in only a handful of patients. STAT5B mediates signaling from the human growth factor receptors such that clinical features include dwarfism, prominent forehead, saddle nose and a high-pitched voice. Since STAT5B is also the primary transcription factor that mediates IL-2 stimulated gene transcription in T cells, most patients also have a marked immunodeficiency, with recurrent varicella virus, herpes virus and Pneumocystis jiroveci infections ( Figure 13-2 ).
In addition to immunodeficiency, most patients also have symptoms suggestive of immune dysregulation including chronic, early-onset diarrhea, eczema and lymphocytic interstitial pneumonitis ( Box 13-5 ). Mice lacking Stat5b have a significant reduction in the number of Foxp3 + T REG cells in thymus and spleen, resulting in splenomegaly and a marked increase of activated T cells in the periphery.
Immunopathogenesis of STAT5B Deficiency
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Growth abnormalities: dwarfism due to growth hormone insensitivity (growth hormone levels normal but insulin-like growth factor 1 levels very low)
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Autoimmunity: IPEX-like enteropathy plus other autoimmunity (particularly pulmonary) as a result of low regulatory T cell numbers
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Immunodeficiency: mild to moderate T and NK cell deficiency associated with recurrent/chronic viral and fungal infections
Genetics and Immunopathogenesis
Two patients have been studied for the effect of STAT5B deficiency on T REG cells. One had a homozygous missense mutation (A630P) and the second had a homozygous nonsense mutation (R152X). Both mutations resulted in markedly reduced or absent STAT5B expression. These patients had significantly fewer CD4 + CD25 high cells than normal controls, FOXP3 expression was decreased and the cells had no in vitro suppressive activity. In addition, decreased CD25 expression (~20% of normal) was observed following T cell activation and is thought to synergize with the underlying STAT5B mutation to effectively abrogate IL-2 signals required for the maintenance of FOXP3 expression and T REG function. Interestingly, signaling pathways required for IL-2 induced expression of other effector molecules, such as perforin, remained intact in STAT5B-deficient T cells.
Diagnosis and Treatment
Diagnosis of STAT5B deficiency is suspected in patients with the overt physical features of dwarfism combined with a significant immunodeficiency. Patients typically have normal serum growth hormone levels but very low insulin-like growth factor 1 (IGF-1) levels. Immunologically, patients generally have low NK cell numbers and modest T cell lymphopenia. Sequencing of the STAT5B gene should be done to confirm the diagnosis.
Treatment of patients with STAT5B deficiency is generally focused on symptomatic therapy and prophylaxis against infections. There are no published reports of bone marrow transplantation (BMT) for STAT5B deficiency although murine studies demonstrate that BMT is curative in mice lacking Stat5a/5b. suggesting that this would correct the immune deficiency and dysregulation but not the growth abnormalities.
Autoimmune Lymphoproliferative Syndrome
Initial reports of patients presenting with lymphadenopathy and hepatosplenomegaly associated with autoimmune cytopenias and increased gammaglobulins were followed 25 years later by a report that identified similar patients also noted to have a marked increase in circulating α/β-TCR + CD3 + CD4 − CD8 − T cells. These α/β double negative T (DNT) cells normally constitute less than 1.5% of peripheral blood lymphocytes in adults ( Figure 13-3 ). The combination of findings led to the suggestion that this could represent the human equivalent to the lpr and gld murine models of autoimmunity.
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