DataSheet_2_Teriflunomide Treatment of Multiple Sclerosis Selectively Modulates CD8 Memory T Cells.pdf

Background and ObjectivesInhibition of de novo pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide.MethodsHigh-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles.ResultsWe found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory TREG follicular helper TFH cell and helper TH cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism.DiscussionOverall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.</p

DataSheet_1_Teriflunomide Treatment of Multiple Sclerosis Selectively Modulates CD8 Memory T Cells.pdf

Background and ObjectivesInhibition of de novo pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide.MethodsHigh-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles.ResultsWe found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory TREG follicular helper TFH cell and helper TH cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism.DiscussionOverall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.</p

Image_2_Teriflunomide Treatment of Multiple Sclerosis Selectively Modulates CD8 Memory T Cells.tif

Background and ObjectivesInhibition of de novo pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide.MethodsHigh-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles.ResultsWe found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory TREG follicular helper TFH cell and helper TH cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism.DiscussionOverall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.</p

Image_1_Teriflunomide Treatment of Multiple Sclerosis Selectively Modulates CD8 Memory T Cells.tif

Background and ObjectivesInhibition of de novo pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide.MethodsHigh-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles.ResultsWe found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory TREG follicular helper TFH cell and helper TH cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism.DiscussionOverall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.</p

Summary of demographic and clinical characteristics of patients analyzed using single HLA-A*0201 antigen coated beads.

<p>Summary of demographic and clinical characteristics of patients analyzed using single HLA-A*0201 antigen coated beads.</p

Principle of the method of identification of antigen-specific B cells.

<p>After co-incubation, lymphocytes, antigen covered beads and the beads’ B cell rosettes are gated based on their forward scatter and side scatter. After exclusion of the DAPI+ cells, B cells and beads-B cell rosettes are identified based on CD19 expression and the beads’ internal fluorochrome. Specificity of B cell recognition is determined by gating on beads and beads’ B cell rosettes (<b>A</b>) or after the identification of the nominal antigen through the use of the unique ratio of the two internal fluorochromes (<b>B</b>). In the latter, for each nominal antigen, a gate that encompassed beads and B cell rosettes is created followed by the identification of the B cells. Frequency of B cells bound to HLA class I of interest is finally evaluated. Bead-based method allows the detection of antigen-specific B cells. (<b>C</b>). An example of the identification of beads, Bead-cell rosette and lymphocyte is shown. After exclusion of dead cells, the use of the marker CD19 allows the identification of B lymphocyte and a mix of beads and BBR. Thanks to the ratio of two fluorochromes, antigen coated on the beads can be then identified. Beads are excluded using the expression of CD19. A Boolean gate is used to assess the frequency of B cells specific of a given antigen within the whole B cell population.</p

Bead-based method allows the detection of antigen-specific B cells.

<p>(<b>A</b>) B cells purified from Tg mice were incubated with human albumin, MOG_1–125 or pp65 coated beads and the frequency of antigen specific B cells was quantified. The B cells were preincubated with soluble human Albumin, MOG_1–125 or pp65 before incubation with MOG_1–125 coated beads. Data are presented as mean ± sem <b>B</b>). B cells purified from Tg mice were preincubated with increasing doses of soluble MOG_1–125 before incubation with MOG_1–125 coated beads. The experiments were repeated 3 times and similar results were obtained.</p

Separation of B cells interacting with nominal antigen and unbound B cells.

<p>Purified B cells were incubated with single HLA class I coated beads (A) or MOG_1–125 coated beads (B) before being subjected to cell separation using an ARIA FACS-sorter (A) or magnet based purification (B). Frequency of B cells interacting with nominal antigens is shown before purification and in the positive and in the negative fraction. One representative out of three experiments with cells from different donors is shown.</p

The interaction between CD19⁺ cells and HLA class I coated beads is not restricted by the HLA class I allele only and BBR are not restricted to the memory compartment.

<p><b>A.</b> CD19⁺ cells were pre-incubated with a cocktail of HLA-A*0201 multimer (HLA-A*0201/MP_58–66, HLA-A*0201/HCw1, HLA-A*0201/pp65) followed by the incubation with HLA Class I coated beads. Frequency of CD19⁺ cells specific of HLA-A*0201 coated beads and of HLA class I coated beads were analyzed before and after coincubation with the cocktail of HLA-A*0201 multimer for healthy volunteers (n = 6) and immunized patients (n = 3). Data are presented as mean ± sem. <b>B.</b> B cells were stained with anti-CD19, anti-CD27 and anti-IgD antibodies prior to incubation with single HLA coated beads. Phenotype of B cells and BBR were analyzed based on the expression of CD27 and IgD. 4 populations were identified (CD27⁻IgD⁺, naïve B cells; CD27⁺IgD⁺, non-switched memory B cells; CD27⁺IgD⁻, switched memory B cells; CD27⁻IgD⁻); CD27⁻IgD⁻ (late memory B cells). Representative phenotype of B cells and single HLA class I coated beads is shown as well as a summary of 5 immunized patients (mean).</p

Summary of demographic and clinical characteristics of patients analyzed using single HLA class I antigen coated beads.

<p>Values are median (min-max) for continuous variables or number of patients (%) for categorical variables. *one missing data and ¶ more than one missing data. Anti-class I Ab analysis, using Luminex HD, was performed for all but 1 patient.</p