Tuure Kinnunen

Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells

Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans.

CVID-associated TACI mutations affect autoreactive B cell selection and activation

Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6-expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications.

Specific peripheral B cell tolerance defects in patients with multiple sclerosis

Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. B cells have recently emerged as major contributors to disease pathogenesis, but the mechanisms responsible for the loss of B cell tolerance in patients with MS are largely unknown. In healthy individuals, developing autoreactive B cells are removed from the repertoire at 2 tolerance checkpoints during early B cell development. Both of these central and peripheral B cell tolerance checkpoints are defective in patients with rheumatoid arthritis (RA) and type 1 diabetes (T1D). Here, we found that only the peripheral, but not the central, B cell tolerance checkpoint is defective in patients with MS. We show that this specific defect is accompanied by increased activation and homeostatic proliferation of mature naive B cells. Interestingly, all of these MS features parallel defects observed in FOXP3-deficient IPEX patients, who harbor nonfunctional Tregs. We demonstrate that in contrast to patients with RA or T1D, bone marrow central B cell selection in MS appears normal in most patients. In contrast, patients with MS suffer from a specific peripheral B cell tolerance defect that is potentially attributable to impaired Treg function and that leads to the accumulation of autoreactive B cell clones in their blood.

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

Background The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts.

Animal‐derived lipocalin allergens exhibit immunoglobulin E cross‐reactivity

Background Although knowledge of the IgE cross‐reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross‐reactivity of animal allergens is poorly known.

Mammalian lipocalin allergens – insights into their enigmatic allergenicity

Most of the important mammal‐derived respiratory allergens, as well as a milk allergen and a few insect allergens, belong to the lipocalin protein family. As mammalian lipocalin allergens are found in dander, saliva and urine, they disperse effectively and are widely present in the indoor environments. Initially, lipocalins were characterized as transport proteins for small, principally hydrophobic molecules, but now they are known to be involved in many other biological functions. Although the amino acid identity between lipocalins is generally at the level of 20–30%, it can be considerably higher. Lipocalin allergens do not exhibit any known physicochemical, functional or structural property that would account for their allergenicity, that is, the capacity to induce T‐helper type 2 immunity against them. A distinctive feature of mammalian lipocalin allergens is their poor capacity to stimulate the cellular arm of the human or murine immune system. Nevertheless, they induce IgE production in a large proportion of atopic individuals exposed to the allergen source. The poor capacity of mammalian lipocalin allergens to stimulate the cellular immune system does not appear to result from the function of regulatory T cells. Instead, the T cell epitopes of mammalian lipocalin allergens are few and those examined have proved to be suboptimal. Moreover, the frequency of mammalian lipocalin allergen‐specific CD4+ T cells is very low in the peripheral blood. Importantly, recent research suggests that the lipocalin allergen‐specific T cell repertoires differ considerably between allergic and healthy subjects. These observations are compatible with our hypothesis that the way CD4+ T‐helper cells recognize the epitopes of mammalian lipocalin allergens may be implicated in their allergenicity. Indeed, as several lipocalins exhibit homologies of 40–60% over species, mammalian lipocalin allergens may be immunologically at the borderline of self and non‐self, which would not allow a strong anti‐allergenic immune response against them.

T Cell Epitope-Containing Peptides of the Major Dog Allergen Can f 1 as Candidates for Allergen Immunotherapy

One prerequisite for developing peptide-based allergen immunotherapy is knowing the T cell epitopes of an allergen. In this study, human T cell reactivity against the major dog allergen Can f 1 was investigated to determine peptides suitable for immunotherapy. Seven T cell epitope regions (A–G) were found in Can f 1 with specific T cell lines and clones. The localization of the epitope regions shows similarities with those of the epitopes found in Bos d 2 and Rat n 1. On average, individuals recognized three epitopes in Can f 1. Our results suggest that seven 16-mer peptides (p15–30, p33–48, p49–64, p73–88, p107–122, p123–138, and p141–156), each from one of the epitope regions, show widespread T cell reactivity in the population studied, and they bind efficiently to seven HLA-DRB1 molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominant in Caucasian populations. Therefore, these peptides are potential candidates for immunotherapy of dog allergy.

Allergen-specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4+ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4+CD45RO+) and naïve (CD4+CD45RA+) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.

The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes.

Background Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing.

Circulating CXCR5+PD-1+ICOS+ Follicular T Helper Cells Are Increased Close to the Diagnosis of Type 1 Diabetes in Children With Multiple Autoantibodies

Although type 1 diabetes (T1D) is primarily perceived as a T cell–driven autoimmune disease, islet autoantibodies are the best currently available biomarker for autoimmunity and disease risk. These antibodies are produced by autoreactive B cells, the activation of which is largely dependent on the function of CD4+CXCR5+ follicular T helper cells (Tfh). In this study, we have comprehensively characterized the Tfh- as well as B-cell compartments in a large cohort of children with newly diagnosed T1D or at different stages of preclinical T1D. We demonstrate that the frequency of CXCR5+PD-1+ICOS+–activated circulating Tfh cells is increased both in children with newly diagnosed T1D and in autoantibody-positive at-risk children with impaired glucose tolerance. Interestingly, this increase was only evident in children positive for two or more biochemical autoantibodies. No alterations in the circulating B-cell compartment were observed in children with either prediabetes or diabetes. Our results demonstrate that Tfh activation is detectable in the peripheral blood close to the presentation of clinical T1D but only in a subgroup of children identifiable by positivity for multiple autoantibodies. These findings suggest a role for Tfh cells in the pathogenesis of human T1D and carry important implications for targeting Tfh cells and/or B cells therapeutically.