T. Petteri Arstila

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.

The Repertoires of Circulating Human CD8+ Central and Effector Memory T Cell Subsets Are Largely Distinct

Memory T cells are divided into central and effector subsets with distinct functions and homing capabilities. We analyzed the composition and dynamics of the CD8(+) T cell repertoire of these subsets within the peripheral blood of four healthy individuals. Both subsets had largely distinct and autonomous TCRbeta repertoires. Their composition remained stable over a 9 month period, during which no cell passage between these subsets was detected despite important size variation of several clones. In one donor, four out of six TCRbeta clonotypes specific for the influenza A virus were detected in the central subset only, while the two others were shared. Altogether, these observations suggest that most effector memory T cells may not have derived from the central memory subset.

Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

Summary Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

Long-lived Memory T Lymphocyte Responses After Hantavirus Infection

Puumala virus (PUUV) is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS), which is an important public health problem in large parts of Europe. We examined the memory cytolytic T lymphocyte (CTL) responses in 13 Finnish individuals who had HFRS between 1984 and 1995. In seven of these donors, we detected virus-specific CTL responses against the PUUV nucleocapsid (N) protein after in vitro stimulation with PUUV. Six novel CD8+ CTL epitopes were defined on the N protein and were found to be restricted by various HLA alleles including A2, A28, B7, and B8. This is the first demonstration of PUUV-specific CTL responses in humans, and the first identification of CTL epitopes on PUUV. In addition, this study provides one of the few characterizations of a human antiviral memory T cell response, without the complicating issues of virus persistence or reinfection. Interferon (IFN)-γ ELISPOT analysis showed that memory CTL specific for these epitopes were present at high frequency in PUUV-immune individuals many years after acute infection in the absence of detectable viral RNA. The frequencies of PUUV-specific CTL were comparable to or exceeded those found in other viral systems including influenza, EBV and HIV, in which CTL responses may be boosted by periodic reinfection or virus persistence.

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.

IL-7 dysregulation and loss of CD8+ T cell homeostasis in the monogenic human disease autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.

Autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is a monogenic autoimmune disease that is caused by mutations in the AIRE gene. Murine studies have linked AIRE to thymocyte selection and peripheral deletional tolerance, but the pathogenesis of the human disease remains unclear. In this study, we show that APECED patients have elevated IL-7 levels and a drastically decreased expression of IL-7R on CD8+ T cells. This is associated with increased proliferation and a decreased expression of the negative TCR regulator CD5 in the CD45RO− subset. The CD45RO− cells also display oligoclonal expansions, decreased expression of the lymph node homing factors CCR7 and CD62L, and increased expression of perforin, consistent with the accumulation of highly differentiated effector cells. The CD45RO−CCR7+CD8+ population of cells with markers characteristic of naive phenotype is also skewed, as shown by decreased expression of CD5 and increased expression of perforin. The putative CD31+ recent thymic emigrant population is likewise affected. These data are consistent with IL-7 dysregulation inducing a decreased threshold of TCR signaling and self-antigen–driven proliferation, probably in synergy with the failed thymic selection. The resultant loss of CD8+ T cell homeostasis is likely to play a significant role in the pathogenesis of APECED. Our findings may also hold lessons for other diseases in which the IL-7–IL-7R pathway has emerged as a risk factor.

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.

The FOXP3 + subset of human CD4 + CD8 + thymocytes is immature and subject to intrathymic selection

FOXP3, believed to be the regulatory T (Treg)‐cell determining factor, is already expressed at the CD4+CD8+ thymocyte stage, but there is disagreement whether these cells are the precursors of mature CD4+CD8− Treg cells. Here, we provide a quantitative analysis of FOXP3 expression in the human thymus. We show that a subset of CD4+CD8+ cells already expressed as much FOXP3 as the FOXP3+ CD4+CD8− cells, and like mature Treg cells were CD127low. In contrast to earlier data, CD8+CD4− thymocytes expressed significantly lower levels of FOXP3 than either the CD4+CD8+ or CD4+CD8− subsets. The CD4+CD8+ double‐positive cells also expressed recombination‐activating gene‐2, suggesting that they were still immature. Although the FOXP3+ double‐positive cells are thus putatively the precursors of the mature CD4+CD8−FOXP3+ subset, their frequency did not predict the frequency of more mature Treg cells, and analysis of T‐cell antigen receptor repertoire showed clear differences between the two subsets. Although these data do not rule out an independent CD4+CD8+ Treg cell subset, they are consistent with a model of human Treg cell development in which the upregulation of FOXP3 is an early event, but the first FOXP3+ population is still immature and subject to further selection. The upregulation of FOXP3 may thus not be the final determining factor in the commitment of human thymocytes to the Treg cell lineage.

A functional complement system is required for normal T helper cell differentiation.

Complement is a fundamental part of the innate immune system, and also modulates B cell responses. Its effects on T cells, however, are less well studied. Here we have studied antigen-specific T cell responses in C3-knockout (C3-KO) C57BL/6 mice. The animals were immunized with ovalbumin (OVA) in complete Freunds adjuvant, which favors T helper 1 (Th1)-type responses. Splenic lymphocytes from C3-KO mice proliferated less in response to OVA stimulation than splenocytes from control wild type (WT) mice. The response in the C3-KO mice was also qualitatively different. The expression of Th1 lineage determining transcription factor T-bet was decreased in OVA-stimulated splenocytes, and the induction of Th1-associated IgG subclasses impaired. In WT mice T cell proliferation in response to OVA was positively correlated with antigen-specific IgG2a and IgG3 levels. In C3-KO mice the proliferative response correlated with antigen-specific IgE levels, consistent with Th2 deviation. The expression of Th1-inducing cytokines IL-12 and IFN-γ was also decreased in the collecting lymph nodes in the C3-KO mice after immunization. Our results show that the complement system and its component C3 participate in the regulation of T cell responses, and that complement function is required for normal T helper cell differentiation.