Follicular regulatory T cells (T_FR) have been extensively characterized in mice and participate in germinal center responses by regulating the maturation of B cells and production of (auto)antibodies. We report that circulating T_FR are phenotypically distinct from tonsil-derived T_FR in humans. They have a lower expression of follicular markers, and display a memory phenotype and lack of high expression of B cell lymphoma 6 and ICOS. However, the suppressive function, expression of regulatory markers, and FOXP3 methylation status of blood T_FR is comparable with tonsil-derived T_FR. Moreover, we show that circulating T_FR frequencies increase after influenza vaccination and correlate with anti-flu Ab responses, indicating a fully functional population. Multiple sclerosis (MS) was used as a model for autoimmune disease to investigate alterations in circulating T_FR. MS patients had a significantly lower frequency of circulating T_FR compared with healthy control subjects. Furthermore, the circulating T_FR compartment of MS patients displayed an increased proportion of Th17-like T_FR. Finally, T_FR of MS patients had a strongly reduced suppressive function compared with healthy control subjects. We conclude that circulating T_FR are a circulating memory population derived from lymphoid resident T_FR, making them a valid alternative to investigate alterations in germinal center responses in the context of autoimmune diseases, and T_FR impairment is prominent in MS.

The disease course of multiple sclerosis (MS) varies greatly between individuals, making prognosis almost impossible. We previously discovered that cytotoxic CD4+ T cells (CD4+ CTL), identified by the loss of CD28, are able to migrate to sites of inflammation, to contribute to tissue damage and to correlate with inflammation, demyelination and disability in an animal model for MS. We hypothesize that CD4+ CTL drive progression of MS and have prognostic value. The percentage of CD4+CD28null T cells was measured in the blood of 176 patients with relapsing-remitting MS (=baseline). Multimodal evoked potentials (EP) combining information on motoric, visual and somatosensoric EP, as well as Kurtzke expanded disability status scale (EDSS) were used as outcome measurements at baseline and after 3 and 5 years. The baseline CD4+CD28null T cell percentage is associated with EP (P=0.003), indicating a link between these cells and disease severity. In addition, the baseline CD4+CD28null T cell percentage has a prognostic value since it is associated with EP after 3 years (P=0.005) and with EP and EDSS after 5 years (P=0.008 and P=0.003). This study provides the first direct link between the presence of CD4+ CTL and MS disease severity, as well as its prognostic value. We elaborate on two research perspectives: 1° investigating strategies to block or reverse pathways in the formation of these cells resulting in new treatments that slow down MS disease progression, 2° including immunophenotyping in prediction modelling studies to aim for personalized medicine.

The long term effects of fingolimod, an oral treatment for relapsing-remitting (RR) multiple sclerosis (MS), on blood circulating B and T cell subtypes in MS patients are not completely understood. This study describes for the first time the longitudinal effects of fingolimod treatment on B and T cell subtypes. Furthermore, expression of surface molecules involved in antigen presentation and costimulation during fingolimod treatment are assessed in MS patients in a 12 month follow-up study. Using flow cytometry, B and T cell subtypes, and their expression of antigen presentation, costimulation and migration markers were measured during a 12 month follow-up in the peripheral blood of MS patients. Data of fingolimod-treated MS patients (n = 49) were compared to those from treatment-naive (n = 47) and interferon-treated (n = 27) MS patients. In the B cell population, we observed a decrease in the proportion of non class-switched and class-switched memory B cells (p<0.001), both implicated in MS pathogenesis, while the proportion of naive B cells was increased during fingolimod treatment in the peripheral blood (PB) of MS patients (p<0.05). The remaining T cell population, in contrast, showed elevated proportions of memory conventional and regulatory T cells (p<0.01) and declined proportions of naive conventional and regulatory cells (p<0.05). These naive T cell subtypes are main drivers of MS pathogenesis. B cell expression of CD80 and CD86 and programmed death (PD) -1 expression on circulating follicular helper T cells was increased during fingolimod follow-up (p<0.05) pointing to a potentially compensatory mechanism of the remaining circulating lymphocyte subtypes that could provide additional help during normal immune responses. MS patients treated with fingolimod showed a change in PB lymphocyte subtype proportions and expression of functional molecules on T and B cells, suggesting an association with the therapeutic efficacy of fingolimod.

CD4+CD28− T cells arise through repeated antigenic stimulation and are present in diseased tissues of patients with various autoimmune disorders, including multiple sclerosis (MS). These cells are believed to have cytotoxic properties that contribute to the pathogenic damaging of the target organ. Endogenous cues that are increased in the diseased tissue may amplify the activity of CD4+CD28− T cells. In this study, we focused on IL-15, a cytotoxicity-promoting cytokine that is increased in the serum and cerebrospinal fluid of MS patients. Using immunohistochemistry, we demonstrate that IL-15 is mainly produced by astrocytes and infiltrating macrophages in inflammatory lesions of MS patients. Moreover, in vitro transmigration studies reveal that IL-15 selectively attracts CD4+CD28− T cells of MS patients, but not of healthy individuals. IL-15 further induces the expression of chemokine receptors and adhesion molecules on CD4+CD28− T cells, as investigated using flow cytometry, resulting in enhanced migration over a monolayer of human brain endothelial cells. Finally, flow cytometric analyses revealed that IL-15 increases the proliferation and production of GM-CSF, expression of cytotoxic molecules (NKG2D, perforin, and granzyme B), and degranulation capacity of CD4+CD28− T cells. Taken together, these findings indicate that increased peripheral and local levels of IL-15 amplify the pathogenic potential of CD4+CD28− T cells, thus contributing to tissue damage in MS brain lesions.