Optimal response to dimethyl fumarate is mediated by a reduction of Th1‐like Th17 cells after 3 months of treatment

Abstract Aim Dimethyl fumarate (DMF) is one of the most promising therapies for relapsing‐remitting multiple sclerosis (RRMS) patients since it has shown immunomodulatory and neuroprotective effects. However, a percentage of RRMS patients do not exhibit an optimal response to DMF. The objective of this study was to identify early biomarkers of treatment response by analyzing changes in peripheral leukocyte subpopulations directly in whole blood samples. Methods A longitudinal and prospective study analyzing peripheral blood leukocyte subpopulations in 22 RRMS patients before initiating DMF treatment (baseline) and at 1, 3, 6, and 12 months of follow‐up was performed. Differences between no evidence of disease activity (NEDA) and ongoing disease activity (ODA) patients were analyzed. Results The beneficial effect of DMF was associated with a specific depletion of memory CD4+ and CD8+ T lymphocytes and B cells. Importantly, only NEDA patients showed (a) a shift from a pro‐ to an antiinflammatory profile, with an increase of Th2 cells and a decrease of Th1‐like Th17 lymphocytes; and (b) an increase of regulatory CD56bright NK cells. Conclusion The optimal response to DMF is mediated by a shift to antiinflammatory and immunoregulatory profile, which puts forward Th1‐like Th17 lymphocytes as a potential early biomarker of treatment response.


| INTRODUC TI ON
Multiple sclerosis (MS) is a chronic immune-mediated disease characterized by inflammation, demyelination, and axonal damage in the central nervous system (CNS). During the last years, numerous new immunomodulatory drugs have been approved for the treatment of MS patients. From them, delayed-release dimethyl fumarate (DMF) is one of the most promising new treatments for relapsing-remitting MS (RRMS) patients, since it has showed both antiinflammatory and neuroprotective properties. Clinically, DMF treatment reduces the annualized relapse rate (ARR) and the number of new lesions measured by magnetic resonance imaging (MRI). 1,2 Nowadays, due to the numerous disease-modifying therapies (DMT) available for MS patients, the selection of the proper treatment for each patient is getting more difficult. Currently, the decision is based on the balance between the potential clinical benefits and the adverse effects of each treatment. However, it is known that a percentage of patients exhibit a suboptimal clinical response to some DMT. Research groups around the world have been investigating the mechanism of action of each DMT, trying to identify molecules or biomarkers of treatment response. Dimethyl fumarate mediates immunomodulatory as well as antioxidative and cytoprotective effects through the inhibition of the transcription factor nuclear factor kappa b (NF-κB) and promoting the activation of the nuclear factor-erythroid 2-related factor (Nfr2) transcriptional pathway. [3][4][5][6] Studies focused on the mechanism of action of DMF demonstrated that the treatment induces lymphopenia, selectively depleting memory T and B cells and promoting a shift toward a tolerogenic immune profile. [7][8][9][10][11][12] Specifically, Medina and colleagues reported that DMF induces a change in the ratio between regulatory CD56 bright NK cells and effector TNF-α-producing CD8 + T cells, which is required for the efficacy of the treatment. The authors also suggested that monitorization of leukocyte subpopulations could be a useful tool for early identification of responder and nonresponder patients. 12 In this study, we performed a longitudinal and extensive characterization of whole blood immunophenotype of RRMS patients before and after 1, 3, 6, and 12 months of DMF treatment by flow cytometry. Changes in lymphocyte subpopulations in patients with no evidence of disease activity (NEDA) in comparison with patients with ongoing disease activity (ODA) were analyzed in order to identify early biomarkers of treatment response. This technique is an easy and fast way to directly analyze (without the limitations of leukocyte isolation or cryopreservation) the ex vivo effect of DMF or other treatments in leukocyte subpopulations. Using this protocol, we identified that a reduction of effector memory Th1-like Th17 cells is associated with increased DMF efficacy, which occurs already after 3 months of treatment in NEDA patients and it is maintained until 12 months of treatment. Patients were classified as NEDA (no evidence of disease activity)-defined as the absence of relapses, disability progression, and radiological activity during the follow-up-or ODA-when an increase in clinical and/or radiological activity was reported.

| Patients and study design
Blood samples were obtained from patients at baseline and after 1, 3, 6, and 12 months of treatment, to determine changes in lymphocyte subpopulations related to an optimal clinical response to DMF.
The study protocol was approved by the ethics committee of the Germans Trias i Pujol Hospital, and all the patients gave their informed consent to participate in the study.

| Analysis of peripheral leukocyte subpopulations
For the analysis of the different peripheral blood leukocyte subpopulations, whole blood samples, obtained by standard venipuncture in EDTA tubes, were kept at room temperature and processed within the next 24 hour. Briefly, 100µl of peripheral blood was incubated for 20 minutes at room temperature and protected from light, with the appropriate combination of antibodies (see below) to analyze T-cell, Treg, B-cell, and DC/monocyte/NK-cell subpopulations.
After erythrocyte lysis and cell fixation, samples were washed twice and acquired on a LSR II Fortessa cytometer and analyzed using FACSDiva software (BD Biosciences). Cell markers used to define each leukocyte subpopulations are specified in Table S1. The gating strategy used is based on an international consensus and was previously described by Teniente-Serra and Quirant-Sánchez. 13,14 Leukocyte subpopulations were analyzed by using the following four panels of monoclonal antibodies, previously reported by

| Statistical analysis
Statistical analyses were performed using GraphPad Prism version 6.00 for Windows. Parametric and nonparametric tests were used depending on the normality of the distribution of the variables. Differences between samples obtained at different time points were compared to baseline measurements using the repeated

| Patients
A total of 22 MS patients were enrolled in the study. To determine the effects of DMF related to an optimal response, 21 out of 22 patients were classified as NEDA (n = 15) or ODA (n = 6) patients. One F I G U R E 1 Changes induced by DMF treatment on T-and B-lymphocyte subpopulations. Representation of percentages (%) of central memory (CCR7 + CD45RA − ), effector memory (CCR7 − CD45RA − ), and naïve (CCR7 + CD45RA + ) subpopulations from CD4 + and CD8 + T lymphocytes (A and B, respectively); and % of preswitched memory (CD27 + IgD + ), class-switched (CD27 + IgD − ) and (CD27 − IgD + ) naïve CD19 + B lymphocytes (C), before the treatment (baseline) and after 1, 3, 6 and, 12 months of follow-up. Data are expressed as mean SD. Each dot represents the value of an individual (n = 22). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 patient was excluded from this stratification due to the lack of MRI at 12 months. No differences were found on baseline characteristics between groups, and, as expected, the ODA group showed a higher number of relapses (P = 0.0016) and an increased presence of new T2 lesions (P = 0.0025) after 12 months of follow-up (Table 1).

| Optimal response to DMF is related to a reduction of memory CD4 + T cells
As has been widely described, the analysis of whole blood samples revealed that DMF induced a progressive decrease in the absolute number of lymphocytes (P = 0.0005). This reduction was statistically significant after 6 months of treatment and remained after 12 months of follow-up ( Figure S1A and Table S2).
When the effect of DMF on T cells was investigated, a decrease in the percentage of CD3 + T cells was found (P < 0.0001) following just 3 months of treatment ( Figure S1B). Further analyses revealed that the number of both CD8 + and CD4 + T cells was reduced although CD4 + lymphocytes were decreased in a lesser extent (Table S2). Consequently, the proportion of CD4 + T cells and the ratio of CD4 + /CD8 + T lymphocytes were significantly increased at 6 and 12 months of DMF treatment (P < 0.0001 and P = 0.0001, respectively) ( Figure S1C).  Table S3). These changes were statistically significant at 6 and 12 months of treatment for effector memory CD4 + and CD8 + T cells.
In the case of central memory CD8 + and CD4 + T lymphocytes, differences were found after 3 or 12 months of follow-up, respectively.
Interestingly, when patients were classified as NEDA or ODA, the effects caused by the DMF treatment on CD4 + and CD8 + T-lymphocyte subpopulations were only observed in NEDA patients, but not in ODA patients, with the exception of CD8 + central memory cells, which were reduced in both groups of patients (Figure 2A-D and Table S3). In addition, NEDA patients exhibited a significant reduction of both CD4 + and CD8 + central memory T lymphocytes after 3 months of treatment (P < 0.0001 and P = 0.0021, respectively) ( Figure 2A,C and Table S3).
The analysis of B-cell subsets revealed comparable results to those found in T-cell subpopulations (Tables S4 and S5) Table S5).

| DMF reduces Th1-like Th17 lymphocytes in NEDA patients
When the pro-and antiinflammatory effects of DMF were evaluated ( Figure S2) by analyzing whole blood samples, a reduction of the Th1-like Th17 population was found after 6 and/or 12 months of treatment in central memory and effector memory cells, respectively (P < 0.0001 in both subsets, Figure S3). Importantly, an increase in the percentage of central and effector memory Th2 lymphocytes was also observed (P < 0.0001 and P = 0.0065, respectively). This antiinflammatory effect was significantly relevant after 3 months of treatment for central memory Th2 cells and after 12 months in the case of effector memory Th2 cells (Table S3). Surprisingly, the analysis exhib-  Table S3). Interestingly, differences in the percentage of Th1-like Th17 effector memory cells between NEDA and ODA patients were statistically significant after 3 and 6 months of treatment (P = 0.0202 and P = 0.0373, respectively).
On the other hand, the analysis of memory Treg in the whole group of MS patients showed a progressive reduction of this population, being statistically significant after 3 months of DMF treatment (P = 0.0001; Figure 4G). However, the percentage of activated memory Treg remained stable during the whole observation period ( Figure 4H). Results obtained considering NEDA and ODA patients were similar for both memory Treg and activated Treg subpopulations (Tables S2 and S3).   (Figure 5A,B).  Although etiology of MS still remains unknown, it is considered an immune-mediated disease in which myelin-reactive CD4 + T lymphocytes trigger a complex immune response that leads to inflammation, myelin destruction, and axonal loss. [17][18][19] The main players initiating the autoimmune attack are autoreactive CD4 + Th1 and Th17 cells, which produce pro-inflammatory cytokines, such as IFN-γ and IL-17, respectively. These cells are able to cross the blood-brain barrier and reach the CNS and recognize myelin antigens, secreting pro-inflammatory mediators and activating other immune cells, such as B cells and cytotoxic CD8 + T cells. 20 Consequently, different pharmacological strategies have been developed to abrogate or block these immune cascades.

| D ISCUSS I ON
In this study, we prospectively evaluated changes induced by DMF treatment performing a direct whole blood cytometric analysis. This approach provided a deep analysis of the adaptive and innate immune response, being able to reproduce most of the DMF effects reported during last years.
As described, DMF reduced the absolute number of circulating lymphocytes, affecting more extensively CD8 + cytotoxic T cells 8,21 and therefore increasing the CD4 + /CD8 + T-cell ratio. 9 whether this reduction is related to a decrease of pro-inflammatory Th1 and/or Th17 Treg. 26,27 Regarding discrepancies with other studies reporting a reduction on Th1 and Th17 cells and no alteration or even expansion of Treg, 8,9,12 they might be caused by the use in those studies of isolated, cryopreserved, and stimulated PBMC (since it has been reported that these procedures can modify the expression of chemokine receptors). 28

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.