Eomesodermin expression in CD4+T‐cells associated with disease progression in amyotrophic lateral sclerosis

Abstract Aim To clarify the role of Eomesodermin (EOMES) to serve as a disease‐relevant biomarker and the intracellular molecules underlying the immunophenotype shifting of CD4+T subsets in amyotrophic lateral sclerosis (ALS). Methods The derivation and validation cohorts included a total of 148 ALS patients and 101 healthy controls (HCs). Clinical data and peripheral blood were collected. T‐cell subsets and the EOMES expression were quantified using multicolor flow cytometry. Serum neurofilament light chain (NFL) was measured. In 1‐year longitudinal follow‐ups, the ALSFRS‐R scores and primary endpoint events were further recorded in the ALS patients of the validation cohort. Results In the derivation cohort, the CD4+EOMES+T‐cell subsets were significantly increased (p < 0.001). EOMES+ subset was positively correlated with increased serum NFL levels in patients with onset longer than 12 months. In the validation cohort, the elevated CD4+EOMES+T‐cell proportions and their association with NFL levels were also identified. The longitudinal study revealed that ALS patients with higher EOMES expression were associated with higher progression rates (p = .010) and worse prognosis (p = .003). Conclusions We demonstrated that increased CD4+EOMES+T‐cell subsets in ALS were associated with disease progression and poor prognosis. Identifying these associations may contribute to a better understanding of the immunopathological mechanism of ALS.


| INTRODUC TI ON
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of upper and lower motor neurons that leads to paralysis, dysphagia, dysarthria, and eventually respiratory failure. 1 Multiple factors may contribute to ALS susceptibility.Among them, neuroinflammation is one of the most important hallmarks and potential therapeutic targets of ALS. 2 It has been well documented that ALS patients have persistent peripheral inflammation, which may exacerbate the neurodegenerative process.Among them, the CD4 + T-cell counts were considered to play a critical role.CD4 + T-cells were significantly reduced in a cohort of 284 ALS patients from West China, 3 as well as in other reports from Australia and the United States. 4,57][8] Elimination of the peripheral CD4 + T-cells in the transgenic mice accelerated the clinical severity. 9,10A population-based longitudinal cohort study recently demonstrated that several altered T-cell subpopulations were related to the risk of death in ALS patients. 11However, the intracellular molecules underlying the phenotype shifting in CD4 + T-cells among ALS remain largely unelucidated.
3][14][15] The EOMES expression in CD4 + T-cells has been reported to be essential in the induction of chronic neuroinflammation. 16More recently, elevated EOMES-positive T-cells were demonstrated in patients with secondary progressive multiple sclerosis (SPMS), a central nervous system (CNS) demyelinating disease with neurodegenerative pathogenesis.The EOMES-positive T subsets were significantly associated with the disease progression and were able to predict the disease worsening with 80% accuracy. 17More intriguingly, a recent study has noticed a substantially increased proportion of EOMESexpressing cells in the cerebrospinal fluid (CSF) expanded T-cells of ALS patients. 18Although multiple pieces of evidence have hinted at a potential role of EOMES in the pathogenesis of ALS, the EOMES expression in CD4 + T-cells among ALS patients and its potential influence on the disease progression have not been clarified thoroughly.
Thus, we hypothesized that the EOMES expression in CD4 + Tcells was potentially associated with the CD4 + T phenotype shifting and the disease progression among ALS patients.In the current study, we used multicolor flow cytometry to measure the changes in EOMES expression among peripheral CD4 + T subsets and assessed the associations between these subsets with disease progression and serum neurofilament light chain (NFL) in a cohort with ALS.

| Study population
The Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) was used for clinical assessment.King's staging system 20 and ALS-Milano Torino functional staging system (ALS-MiToS) 21 were used for patient grading.Exclusion criteria for both patients and controls were acute or chronic infection, a positive family history of ALS, a previous diagnosis of neurodegenerative disorders, autoimmune diseases, or immunomodulatory medication at blood sampling.In 1-year longitudinal follow-ups, the ALSFRS-R scores and primary endpoint events (including invasive mechanical ventilation, using noninvasive mechanical ventilation over 22 h per day, and death) were further recorded in the validation cohort recruited from Fujian Medical University Union Hospital.A flowchart displaying the study design is presented in Figure S1.The study protocol was approved by the ethics committees of Fudan University Huashan Hospital (2019-603) and Fujian Medical University Union Hospital (2019GZR032).Written informed consent was obtained from each participant.

| Blood sampling and flow cytometry
Peripheral blood was collected using a BD Vacutainer® blood collection tube containing EDTA and immediately sent for flow cytometry analysis.For flow cytometry, a 200-μL aliquot of whole blood was stained with a cocktail of surface markers, which was selected mainly derived from the standardized phenotyping panel by the Human Immunophenotyping Consortium 22 (the antibodies included anti-CD3, anti-CD4, anti-CXCR3, anti-CCR4, anti-CCR6, anti-CCR7, anti-CD45RA, anti-CD25, and anti-CD127).Then, the mixture was lysed using red blood cell lysing buffer (TIANGEN).Subsequently, cells were fixed and permeabilized using eBioscience and stained with anti-EOMES antibody (clone: WD1928; Thermo Fisher Scientific) according to the manufacturer's instructions.
Appropriate fluorescein-conjugated, isotype-matched, irrelevant antibodies were used as isotype controls.The flow cytometry experiments of the derivation cohort were performed using Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed on FlowJo VX Software (FlowJo, LLC).The flow cytometry experiments of the validation cohort were performed using BD FACSCelesta™ Flow Cytometer (BD Life Sciences).

| Serum NFL quantification
Serum NFL levels were quantified with NFL assay kits (Cat No: 103186; Quanterix) on the ultrasensitive single-molecule array (SIMOA) platform according to the manufacturer's instruction.
Calibrators and quality controls were measured in duplicates.All sample measurements were diluted at a 1:4 ratio and performed on a single-run basis.Operators were blinded to the participants' disease status.

| Statistical analyses
Qualitative variables were compared by the chi-square or Fisher's exact test.Quantitative variables for the ALS patients and HCs were summarized with descriptive statistics, such as mean, standard deviation (SD), median, interquartile range (IQR), and proportions.For CD4 + T subsets, proportions less than 0.05% were not taken into statistical analysis.Mann-Whitney tests were performed for comparisons.A receiver operating characteristic (ROC) curve was generated, and the area under the curve (AUC) was calculated to assess the performance of CD4 + T subsets.
The Spearman correlation was used in assessing the associations among the patient's functional status, serum NFL, and immune biomarkers.To explore the association between the EOMES expression and the risk of developing endpoint events, multiple variate COX regression models were adopted and survival curves were constructed.Log-rank test was then applied to assess the differences between the survival curves.Statistical analyses and visualizations were performed using GraphPad Prism version 9 and R version 4.2.2.p-Values were two-tailed with a significant level of 0.05.

| Clinical and demographic features of the study population
The clinical and demographic features are summarized in Table 1

| EOMES was highly expressed in the peripheral CD4 + T-cells derived from ALS patients
The frequencies of CD4 + T subsets are summarized in  2).Moreover, the EOMES expression was enriched in Th1, Th17.1, and Th9 subsets in ALS patients and HCs (Table 2).Meanwhile, the EOMES was preferentially expressed in effector memory subsets (TEMRA and TEM).Moreover, we noticed that for those in subgroups with bulbar onset, the patients have

| Elevated CXCR3 expression in CD4 + EOMES + T-cells differentiates ALS from HCs
The EOMES expression was enriched in Th1, Th17.1, and TEMRA and TEM subsets, which were gated mainly according to the higher expression of CXCR3 and the lower expression of CCR7 or CD27.
Receiver operating characteristic curves were further calculated to assess the predictive performance of the above-mentioned CD4 + T subsets in the derivation cohort.As was shown in Table 3 and Figure 1H, the CXCR3 Then, we compared the patients within 6 months of onsets.In the derivation cohort, 19 ALS patients' onsets were within 6 months.

| EOMES + subsets were correlated with serum NFL levels
Both CD4 + T subsets and EOMES-positive subsets were not correlated with the decreased rate of the ALSFRS-R scores.We assumed that the clinical scale might not be sensitive enough.Thus, we further quantified the patients' serum NFL level to evaluate subclinical motor neuron damage.In line with the previous studies, the NFL levels of the ALS patients were negatively correlated with disease duration and positively correlated with the decreased rate of ALSFRS-R scores per month (ρ = −0.492,p < 0.001 and ρ = 0.494, p < 0.001).The direct linear correlations were not significant among subsets.Previous longitudinal studies have demonstrated that the blood NFL will dynamically increase in the early stage of the disease course (first 6-20 months) and remain relatively stable over times. 25,26Thus, we selected ALS patients with a disease duration of more than 12 months.We observed correlations between EOMES subsets and serum NFL levels.
The EOMES + and CXCR3 + EOMES + subsets showed a tendency of

| EOMES expression was correlated with ALS patients' survival
To explore the relation between the EOMES expression and the prognosis of ALS, we recorded the ALSFRS-R scores and primary endpoint events in the validation cohort recruited from Fujian Medical University Union Hospital in the following year.A total of 45 ALS patients were followed.Except for four patients dropped, 41 patients received follow-up either in the clinic or by telephone.
The most extended follow-up period was 14 months.During the follow-up period, 9 of 45 ALS patients reported endpoint events.
Unfortunately, COX analysis adjusted for age, gender, disease duration, and site of onset did not reveal statistical significance (Table S1).

| DISCUSS ION
In this study, we performed in-depth peripheral CD4 + T profiling in two independent cohorts of ALS patients and corresponding matched healthy controls using multicolor flow cytometry.For the validation cohort, we longitudinally followed the ALS patients recruited from Fujian Medical University Union Hospital.
We found the CD3 + and CD4 + T-cell counts were significantly decreased in ALS patients.9,10 The immunophenotyping of the conventional CD4 + T subsets revealed increased Th1 proportions in ALS patients.Though without significance, the TEMRA and TEM proportions seemed to be increased, while the TCM proportions seemed to be decreased.The immune profile shifting toward a Th1/Th17 cell-mediated pro-inflammatory immune response in ALS patients has been reported recently. 279][30][31] In addition, a significant augmentation of a Th1-associated transcription factor, T-bet, was revealed in the lumbar cord of SOD1 G93A mice at the mRNA level. 32Here, our results further enhanced the former evi- in experimental autoimmune encephalomyelitis mice. 16Recently, such a close association between the increased CD4 + EOMES + Tcells and the progression of the disease has been further verified in a cohort of 105 SPMS patients 17 and even multiple neurodegeneration mouse models. 35The neurodegenerative mechanisms were believed to contribute a lot to the pathogenesis of SPMS.Thus, a similar role of peripheral EOMES expression in neurodegenerative disorders seemed to be reasonable.Here, in our study, we identified that the proportions of CD4  A strength of our study is the relatively large sample sizes and three-stage design compared to previous studies.However, the median ALSFRS-R score in our ALS group was 41 with a median disease duration of 12 months, which represented a group of patients at an early stage.Thus, further studies are needed to see whether the changes in CD4 + T subsets could be safely and smoothly fitted in patients at the later stages.Another limitation of our study is the discrepancy in bulbar onset patients' number between two stages.In addition, although part of our results is consistent with previous studies, there was a possible reverse causality between the changes in CD4 + T subsets and CNS inflammation.Thus, a longitudinal cohort study with in-depth immunophenotyping would be helpful to further elucidate the role of CD4 + T subset changes.Besides, an investigation involving the CNS-infiltrated CD4 + EOMES + T-cells in ALS patients might be helpful.
In summary, we demonstrated altered peripheral CD4 + T subsets and increased EOMES expression in ALS patients.We further demonstrated their associations with disease progression and prognosis.Identifying these associations in ALS may contribute to a better description of the immunopathological features of ALS.Besides, further understanding of the changes in CD4 + T subsets and the critical role of EOMES expression may contribute to the development of potential therapeutics.

F I G U R E 1 | 7 of 11 CHEN
The EOMES + expression in Th subsets in ALS and HC groups.(A) The EOMES expression was significantly elevated in ALS patients' CD4 + T-cells in the derivation cohort.(B) The EOMES expression was significantly higher in ALS patients with bulbar onset in the derivation cohort.(C) An increasing proportion of bulbar onset patients was observed among EOMES expression quarters in the derivation cohort.(D) The EOMES expression was significantly elevated in ALS patients' CD4 + T-cells in the validation cohort.(E) The EOMES expression was significantly higher in ALS patients with bulbar onset in the validation cohort.(F) An increasing proportion of bulbar onset patients was observed among EOMES expression quarters in the validation cohort.(G) The proportion of CXCR3 + EOMES + subset was elevated in ALS patients in both cohorts.(H) Receiver operating characteristic (ROC) curves were generated to evaluate diagnosis abilities of the EOMES in CD4 + T-cells and CXCR3 + EOMES + Th subset in the derivation cohort.(I) ROC curves were generated to evaluate diagnosis abilities of the EOMES in CD4 + T-cells and CXCR3 + EOMES + Th subset in the validation cohort.ALS, amyotrophic lateral sclerosis; EOMES, eomesodermin; HC, healthy control.et al.

F I G U R E 2
The correlations between serum NFL and EOMES + Th subsets.(A-C) The correlations between serum NFL and EOMES subsets in the derivation cohort.(D-F) The correlations between serum NFL and EOMES subsets in the validation cohort.EOMES, eomesodermin; NFL, neurofilament light chain.
dence.Besides, we noticed an unexpected elevation of TEMRA and TEM proportions in ALS patients.Human TEMRA and TEM subsets are memory cells characterized by rapid effector function, containing Th1, Th2, and cytotoxic T lymphocyte (CTL) cells.A higher ratio of CD45RO/RA expression has been revealed in the PBMCs of ALS twins, suggesting an increased central or effector memory T-cell subset.33Moreover, a recent populationbased longitudinal cohort study revealed that the proportion of TEMRA subset in CD4 + T-cells was increased after ALS diagnosis and associated with higher mortality risk.11Here, in concordance with previous studies, we confirmed this increased memory T-cell subset in ALS patients, and further emphasized the critical roles of Th1 and effector memory subsets in the immunopathological process of ALS.Recently, a hot debate has emerged regarding the role of EOMES in human CD4 + T-cell differentiation.34It has been reported that EOMES could favor the phenotype shift of Th17 cells toward non-classic Th1 cells and plays a key role in T helper cell (Th) plasticity. 14It has been claimed in a previous animal study that CD4 + EOMES + T-cells were required for the augmentation of CNS inflammation on adoptive transfer and the induction of chronic stage F I G U R E 3 The survival curve analyses in the longitudinal study.(A) ALS patients with higher baseline median CD4 + EOMES + T proportions were associated with worse prognosis.(B) ALS patients with higher baseline median CD4 + CXCR3 + EOMES + T proportions were associated with worse prognosis.ALS, amyotrophic lateral sclerosis; EOMES, eomesodermin.
gesting a potential relationship between the elevation of peripheral EOMES and the motor neuron damages.More importantly, we, for the first time, identified higher EOMES expression in peripheral CD4 + T-cells may be associated with worse prognosis in our validation cohort follow-up.Our findings were potently supported by two recent reports.One recent animal study identified accumulated EOMES + Th cells in the CNS of ALS and other neurodegenerative mouse models.35Another study using human CSF identified high levels of effector T-cells were enriched in the ALS patients' CSF, which was associated with poor survival.In their single-cell transcriptomics analysis of CSF samples, clonally expanded CD4 + T-cells were characterized by increased EOMES expression.18Combining these findings, we believed that enriched EOMES in CD4 + T-cells, especially in CXCR3-positive subsets, might represent a specific form of T-cells that might migrate into the CNS and further deteriorate the inflammation environment in ALS patients.In this process, the elevated EOMES expression might be a critical hallmark for ALS patients.
The derivation cohort was comprised of 85 ALS patients and 55 age-and gender-matched healthy controls (HCs), who were recruited from two independent neuromuscular referral centers in Fudan University Huashan Hospital and Fujian Medical University Union Hospital, from September 13, 2018, to June 1, 2021.To further validate our results, a validation cohort, including 63 ALS patients and 46 HCs, was recruited from May 16, 2022, to October 19, 2022.The diagnosis of ALS was made by at least two experienced neurologists according to the revised EI Escorial criteria.
+ EOMES + T-cells were significantly increased in ALS patients.Moreover, we found the EOMES expres- + EOMES + subset proportions in the CD4 + T-cells of ALS patients.Our further ROC curve analysis indicated that the CXCR3 + EOMES + subset might be the most potent univariate predictor for ALS, with the biggest AUC estimated at 0.77.