Summary: CD4⁺ CD25^high regulatory T cells (Tregs) of patients with relapsing‐remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single‐cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4⁺ CD25^high FOXP3⁺ T cells and lower FOXP3 protein expression per cell in RR‐MS patients than in SP‐MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)‐β‐treated RR‐MS patients showed restored numbers of FOXP3⁺ Tregs. Furthermore, a higher percentage of CD4⁺ CD25^high FOXP3⁺ Tregs in RR‐MS patients, as compared with controls and SP‐MS patients, expressed CD103 and CD49d, adhesion molecules involved in T‐cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27⁺ CD25^high CD4⁺ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR‐MS patients. Taken together, these data show aberrant FOXP3 expression at the single‐cell level correlated with Treg dysfunction in RR‐MS patients. Our results also suggest that Tregs accumulate in the CSF of RR‐MS patients, in an attempt to down‐regulate local inflammation in the central nervous system.

Immune aging occurs in the elderly and in autoimmune diseases. Recently, IgD⁻CD27⁻ (double negative, DN) and CD21⁻CD11c⁺ (CD21^low) B cells were described as age-associated B cells with proinflammatory characteristics. This study investigated the prevalence and functional characteristics of DN and CD21^low B cells in multiple sclerosis (MS) patients. Using flow cytometry, we demonstrated a higher proportion of MS patients younger than 60 y with peripheral expansions of DN (8/41) and CD21^low (9/41) B cells compared with age-matched healthy donors (1/33 and 2/33, respectively), which indicates an increase in age-associated B cells in MS patients. The majority of DN B cells had an IgG⁺ memory phenotype, whereas CD21^low B cells consisted of a mixed population of CD27⁻ naive, CD27⁺ memory, IgG⁺, and IgM⁺ cells. DN B cells showed similar (MS patients) or increased (healthy donors) MHC-II expression as class-switched memory B cells and intermediate costimulatory molecule expression between naive and class-switched memory B cells, indicating their potential to induce (proinflammatory) T cell responses. Further, DN B cells produced proinflammatory and cytotoxic cytokines following ex vivo stimulation. Increased frequencies of DN and CD21^low B cells were found in the cerebrospinal fluid of MS patients compared with paired peripheral blood. In conclusion, a proportion of MS patients showed increased peripheral expansions of age-associated B cells. DN and CD21^low B cell frequencies were further increased in MS cerebrospinal fluid. These cells could contribute to inflammation by induction of T cell responses and the production of proinflammatory cytokines.

To determine the role of expanded CD4⁺CD28^null T cells in multiple sclerosis and rheumatoid arthritis pathology, these cells were phenotypically characterized and their Ag reactivity was studied. FACS analysis confirmed that CD4⁺CD28^null T cells are terminally differentiated effector memory cells. In addition, they express phenotypic markers that indicate their capacity to infiltrate into tissues and cause tissue damage. Whereas no reactivity to the candidate autoantigens myelin basic protein and collagen type II was observed within the CD4⁺CD28^null T cell subset, CMV reactivity was prominent in four of four HC, four of four rheumatoid arthritis patients, and three of four multiple sclerosis patients. The level of the CMV-induced proliferative response was found to be related to the clonal diversity of the response. Interestingly, our results illustrate that CD4⁺CD28^null T cells are not susceptible to the suppressive actions of CD4⁺CD25⁺ regulatory T cells. In conclusion, this study provides several indications for a role of CD4⁺CD28^null T cells in autoimmune pathology. CD4⁺CD28^null T cells display pathogenic features, fill up immunological space, and are less susceptible to regulatory mechanisms. However, based on their low reactivity to the autoantigens tested in this study, CD4⁺CD28^null T cells most likely do not play a direct autoaggressive role in autoimmune disease.

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.