Eliisa Kekäläinen

A Defect of Regulatory T Cells in Patients with Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a monogenic recessive disease characterized by autoimmunity against multiple tissues, offers a unique possibility to study the breakdown of self-tolerance in humans. It is caused by mutations in the autoimmune regulator gene (AIRE), which encodes a transcriptional regulator. Work using Aire−/− mice suggests that Aire induces ectopic expression of peripheral Ags and promotes their presentation in the thymus. We have explored reasons for the difference between the comparatively mild phenotype of Aire-deficient mice and human APECED patients. We provide evidence that, unlike in the Aire−/− mice, in the patients a key mediator of active tolerance, the CD4+CD25+ regulatory T (Treg) cell subset is impaired. This was shown by significantly decreased expression of FOXP3 mRNA and protein, decreased function, and alterations in TCR repertoire. Also, in the normal human thymus a concentric accumulation of AIRE+ cells was seen around thymic Hassall’s corpuscles, suggesting that in the patients these cells may be involved in the observed Treg cell failure. In Aire−/− mice the expression of FoxP3 was normal and even increased in target tissues in parallel with the lymphocyte infiltration process. Our results suggest that a Treg cell defect is involved in the pathogenesis of APECED and emphasize the importance of active tolerance mechanisms in preventing human autoimmunity.

Human CD8+ T cell memory generation in Puumala hantavirus infection occurs after the acute phase and is associated with boosting of EBV-specific CD8+ memory T cells.

The induction and maintenance of T cell memory is incompletely understood, especially in humans. We have studied the T cell response and the generation of memory during acute infection by the Puumala virus (PUUV), a hantavirus endemic to Europe. It causes a self-limiting infection with no viral persistence, manifesting as hemorrhagic fever with renal syndrome. HLA tetramer staining of PBMC showed that the CD8+ T cell response peaked at the onset of the clinical disease and decreased within the next 3 wk. Expression of activation markers on the tetramer-positive T cells was also highest during the acute phase, suggesting that the peak population consisted largely of effector cells. Despite the presence of tetramer-positive T cells expressing cytoplasmic IFN-γ, PUUV-specific cells producing IFN-γ in vitro were rare during the acute phase. Their frequency, as well as the expression of IL-7Rα mRNA and surface protein, increased during a follow-up period of 6 wk and probably reflected the induction of memory T cells. Simultaneously with the PUUV-specific response, we also noted in seven of nine patients an increase in EBV-specific T cells and the transient presence of EBV DNA in three patients, indicative of viral reactivation. Our results show that in a natural human infection CD8+ memory T cells are rare during the peak response, gradually emerging during the first weeks of convalescence. They also suggest that the boosting of unrelated memory T cells may be a common occurrence in human viral infections, which may have significant implications for the homeostasis of the memory T cell compartment.

Regulatory T cell defect in APECED patients is associated with loss of naive FOXP3(+) precursors and impaired activated population.

The pathogenetic mechanisms of organ-specific autoimmune diseases remain obscured by the complexity of the genetic and environmental factors participating in the breakdown of tolerance. A unique opportunity to study the pathogenesis of human autoimmunity is provided by autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare inherited autoimmune disease caused by mutations in Autoimmune Regulator (AIRE) gene. Loss of AIRE function disrupts the deletion of autoreactive T cells and impairs the suppressive function of regulatory T (Treg) cells. Here we show by multiparameter flow cytometry that in healthy controls the peripheral naive Treg cell subset forms a slowly dividing, persistent reservoir of recent thymic emigrants (RTEs). In APECED patients the RTE Treg cells show accelerated turnover and shift to the activated pool and the RTE reservoir is depleted. Moreover, the activated Treg cell population in the patients expresses significantly less Forkhead box protein P3 (FOXP3) than in the healthy controls, consistent with the impairment of peripheral activation. Our results indicate that in addition to their thymic effects, loss-of-function mutations in AIRE disrupt the peripheral homeostasis and activation of Treg cells. This may synergize with failed negative selection to cause APECED.

Cutting edge: human CD4-CD8- thymocytes express FOXP3 in the absence of a TCR.

The best candidate for regulatory T (Treg) cell lineage-determining factor is currently the Forkhead box transcription factor FOXP3. FOXP3 up-regulation has been linked to TCR-mediated signals, and in mice the abrogation of TCR expression or signals also prevents FoxP3 expression. In contrast, the TCR dependence of human FOXP3 is assumed but not established. In this study we show on a single cell level that 1.4% (range 0.1–3.8%) of CD4−CD8− thymocytes in healthy humans express FOXP3, two thirds of them without any detectable αβ TCR. These TCR−FOXP3+ cells were mostly CD25− and did not express γδ TCR or B cell, NK cell, or monocyte-associated markers. Like mature Treg cells, they were mostly CD2+CD127low and expressed cytoplasmic CTLA-4. Our results suggest that in immature human thymocytes the expression of FOXP3 precedes surface TCR, in which case TCR-mediated signals cannot be responsible for the thymic up-regulation of FOXP3.

γδ T cells develop independently of Aire

Mutations in the transcriptional regulator Aire disrupt thymic alphabeta T cell selection, causing in humans Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). However, it is not known whether Aire is needed for normal gammadelta T cell development. We show that Aire(-/-) mice have a normal frequency of gammadelta T cells, with TCR repertoire comparable to that of wild-type mice, and normal amount of TCR Cdelta mRNA in ileum and skin. gammadelta T cells did not express increased amounts of CD25 or display hyperproliferation, and were not involved in pathological salivary gland infiltrates. Lastly, the frequency of circulating gammadelta T cells was similar in APECED patients and healthy controls. These data indicate that gammadelta T cells develop independently of Aire and are unlikely to have a significant pathogenetic or protective role in APECED. The antigens responsible for gammadelta and alphabeta T cell selection are thus probably largely different.

T cells expressing two different T cell receptors form a heterogeneous population containing autoreactive clones

During T cell development both alleles of the T cell receptor (TCR) alpha locus are rearranged. As a result, a sizeable proportion of T cells can express two distinct TCRs, but the functional significance of this phenomenon remains controversial. Studies on transgenic mice with two TCRs have focused on the risk of immunopathology that such cells may pose, while some have suggested that most dual-specific T cells are nonfunctional or even protective. We tracked the fate and TCR repertoire of single- and dual-specific T cells within a normal polyclonal population undergoing lymphopenia-induced proliferation, a setting which has been shown to cause immunopathology and autoimmunity. After the expansion the repertoire of dual-specific T cells had become highly biased, with both prominent clonal expansions and the complete disappearance of other clones. Our results suggest that the normal repertoire of dual-specific T cells contains both nonfunctional cells and a small, 5% fraction of clones which display a much higher than average affinity to antigens normally tolerated as harmless. This heterogeneity may also help in reconciling some of the earlier, conflicting results.

In vivo analysis of helper T cell responses in patients with autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy provides evidence in support of an IL-22 defect.

Abstract Autoimmune polyendocrinopathy – candidiasis – ectodermal dystrophy (APECED) is caused by mutations in the Autoimmune regulator (AIRE) gene and is associated with neutralizing anti-cytokine autoantibodies. We have used an in vivo challenge model to analyze antigen-specific CD4+ T cell responses. Bacille Calmette–Guérin (BCG)-vaccinated patients and controls were injected tuberculin intradermally, skin blisters were induced by suction on the indurations and on unexposed skin, and the infiltrating cells harvested. The patients had a quantitatively normal CD4+ T cell response and no significant abnormalities in the expression of T helper type (Th) 1- or Th2-related genes. The expression of interleukin (IL)-22, in contrast, was lower in the patients. Two patients, both with a pre-existing ocular keratopathy, experienced a relapse of keratoconjunctivitis, suggesting a possible immunological basis for this APECED component. Our in vivo data are compatible with a selective IL-22 defect in the activated CD4+ T cells of APECED patients, affecting also unexposed skin in steady-state conditions.

Autoimmunity, Not a Developmental Defect, is the Cause for Subfertility of Autoimmune Regulator (Aire) Deficient Mice

Autoimmune regulators (AIRE) best characterized role is in the generation immunological tolerance, but it is also involved in many other processes such as spermatogenesis. Loss‐of‐function mutations in AIRE cause a disease called autoimmune polyendocrinopathy, candidiasis and ectodermal dystrophy (APECED; also called autoimmune polyendocrinopathy syndrome type 1, APS‐1) that is dominated by various autoimmune manifestations, mainly endocrinopathies. Both patients with APECED and Aire−/− mice suffer from varying levels of infertility, but it is not clear if it is a result of an autoimmune tissue damage or more of a developmental defect. In this study, we wanted to resolve whether or not the reduced fertility of Aire−/− mice is dependent on the adaptive immune system and therefore a manifestation of autoimmunity in these mice. We generated lymphopenic mice without Aire expression that were devoid of the autoimmune manifestations previously reported in immunocompetent Aire−/− mice. These Aire−/−Rag1−/− mice regained full fertility. This confirms that the development of infertility in Aire−/− mice requires a functional adaptive immune system. We also show that only the male Aire−/− mice are subfertile, whereas Aire−/− females produce litters normally. Moreover, the male subfertility can be adoptively transferred with lymphocytes from Aire−/− donor mice to previously fertile lymphopenic Aire−/− recipients. Our data show that subfertility in Aire−/− mice is dependent on a functional adaptive immune system thus confirming its autoimmune aetiology.

Defective Central Tolerance in Aire-Deficient Mice is not Sufficient to Induce Symptomatic Autoimmunity During Lymphopenia-Induced T cell Proliferation

Transcriptional regulator autoimmune regulator (AIRE) controls thymic negative selection but it is also expressed in secondary lymphoid organs. The relative contribution of AIRE’s central and peripheral function to the maintenance of tolerance is unclear. We transferred mature lymphocytes from Aire−/− or wild‐type donors to Aire+/+ lymphopenic recipients, which allowed us to gauge the autoreactivity inherent in the cells originating in an Aire−/− thymus. In the ensuing lymphopenia‐induced proliferation (LIP), the recipients of cells from Aire−/− showed definite T cell hyperproliferation and developed autoantibodies at a higher frequency than the recipients of wild‐type cells. However, neither of the recipient groups developed clinical symptoms, and pathological tissue infiltrates were also absent. The recipients of Aire−/− cells showed hyperproliferation and increased accumulation of regulatory T cells (Tregs), especially in tissues susceptible to inflammation triggered by LIP. These data are consistent with the view that T cells developing in the absence of Aire are autoreactive. However, overt autoimmunity was prevented, most likely by the suppressive function of Treg cells in the Aire‐sufficient recipients. Our results support the importance of the peripheral AIRE expression in the maintenance of immunological tolerance.

Expanded CD4+ Effector/Memory T Cell Subset in APECED Produces Predominantly Interferon Gamma

PurposeAutoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is a rare human autoimmune disorder caused by mutations in the AIRE (autoimmune regulator) gene. Loss of AIRE disrupts thymic negative selection and gives rise to impaired cytotoxic and regulatory T cell populations. To date, CD4+ T helper (Th) cells remain little studied. This study aims to elucidate their role in APECED pathogenesis.MethodsTh cells were explored in ten APECED patients and ten healthy controls using cell culture assays, multiparameter flow cytometry, and transcriptome analysis.ResultsThe proportions of effector/memory populations were increased while the fraction of naive cells was diminished. The naive population was abnormally activated, with an increased number of cells expressing characteristic Th1, Th2, and Th17 cytokines. No clear deviation to any Th subclass was observed, but transcriptome analysis suggested abnormalities in the Th1 cytokine interferon gamma (IFN-γ) pathway and flow cytometry showed that INF-γ had the highest expression. The augmented INF-γ signaling may promote the function of the putative pathogenic CD8+ cytotoxic population in the patients. In addition, the frequency of CD4+ recent thymic emigrants (RTEs) was decreased in the patients, and RTEs also contained cytokine-producing cells at an increased frequency.ConclusionThese data reveal abnormalities in the Th population and suggest that they may in part be traced to premature activation already in the thymus.