{"paper_id":"378e278c-83eb-4d50-bda5-e9c69597de87","body_text":"Characteristics of peripheral blood lymphocyte subsets in patients with neuromyelitis optica | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characteristics of peripheral blood lymphocyte subsets in patients with neuromyelitis optica Shi Qi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7291609/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Neuromyelitis spectrum disorder (NMOSD) is inflammatory disease of the central nervous system (CNS), mainly affecting the optic nerve and/or spinal cord, and characterized by the presence of aquaporin-4 immunoglobulin G antibody (AQP4-IgG) in the serum. The role and activity of different lymphocyte subsets are crucial for understanding the immune mechanisms and pathogenesis of NMOSD. This study aimed to analyze the levels of lymphocyte subsets in NMOSD patients. Methods: 22 NMOSD patients and 22 individuals without autoimmune diseases were enrolled. Lymphocyte subsets were measured in both groups, including CD3 + cells, CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD19 + B cells, CD3- CD(16/56) + natural killer cells (NK cells), and CD4+/CD8 + ratio, along with the routine peripheral blood lymphocyte percentage. The data were analyzed using SPSS 22.0. Results: The total lymphocyte percentage in routine peripheral blood tests were significantly lower in the NMOSD group compared to controls (p < 0.05). Lymphocyte subset analysis revealed the NMOSD group had a significantly higher proportion of CD3 + CD8 + T cells compared to controls. Additionally, the CD4+/CD8 + T cell ratio was significantly lower in the NMOSD group. No significant differences were observed in the proportions of CD3 + cells, CD3 + CD4 + T cells, CD19 + B cells, or CD3-CD56/16 + NK cells between groups. The total lymphocyte percentage showed a positively correlated with the disease duration(r² = 0.2529, P = 0.017). Conclusion: This study suggests that T cell levels, and CD4/CD8 ratio are connected with the occurrence and development of NMOSD. We observed a reduced total lymphocyte percentage in routine blood tests, an elevated proportion of CD3 + CD8 + T cells and a decreased CD4+/CD8 + T cell ratio compared to controls. These immunological changes show associations with NMOSD status, with the total lymphocyte percentage positively correlating with disease duration. Further investigation of these immune parameters may offer potential avenues for monitoring disease progression and developing adjunctive therapeutic strategies for NMOSD. neuromyelitis optica (NMO) NMO spectrum disorder (NMOSD) autoimmune inflammation aquaporin-4 immunoglobulin G antibody (AQP4-IgG) lymphocyte subsets Figures Figure 1 Figure 2 Introduction Devic’s disease, also known as neuromyelitis spectrum disorder (NMOSD), is a rare inflammatory condition affecting the central nervous system (CNS). It is marked by an autoimmune attack primarily targeting the optic nerve and/or the spinal cord. Population and epidemiological studies indicate that the incidence of NMOSD is relatively low, at approximately 0.3 to 4.4 cases per 100,000 people. NMOSD accounts for a higher proportion among Asian patients with demyelinating disease, ranging between 20% and 48%1. Most individuals with NMOSD are aged 35 to 45 age bracket, though the condition can also affect both children and older adults. With the identification of the AQP4-IgG antibody, which targets aquaporin-4, a key water channel in the CNS, NMOSD is increasingly recognized as a distinct disorder from multiple sclerosis2. It has a high recurrence rate and leads to high rates of disability. Prognostic studies indicate that without intervention, patients with NMOSD face a 58–83% chance of developing motor dysfunction within five years3,4. Current management strategies for NMOSD primarily involve the use of conventional immunosuppressive and immunomodulatory medications. However, more than 30% of NMOSD patients are at risk of recurrence and deterioration. The pathological mechanism of NMOSD is complex, but recently, there have been advances in treatment options, such as eculizumab, satralizumab, etc. In the acute stage, treatments typically involve hormones and plasma exchange, whereas long-term immunosuppressants, particularly those that target B cells, are employed to prevent relapse, which significantly improves the long-term quality of life of patients5. There are several hypotheses on the immunopathogenesis of NMOSD, including the generation of anti-AQP4 antibodies, elevated activity of inflammatory B cell and plasmablasts, dysfunctional B cell tolerance checkpoints, reduced regulatory B cell activity, and the disruption of B cell anergy6. B cells are crucial in the pathogenesis of NMOSD, exerting their influence through various mechanisms. Initially, B cells differentiate into plasmablasts and plasma cells, which produce the disease-causing AQP4-IgG7. Additionally, B cell release inflammatory cytokines, such as IL-6, which can trigger both harmful and inflammatory immune responses. Furthermore, B cells can function as antigen-presenting cells, facilitating the maturation and activation of autoreactive T cells6,7. Lastly, the depletion and subsequent restoration of B cells aids in the recovery of the number and functionality of regulatory B cells8. Recent studies have shown that in addition to B lymphocyte-mediated humoral immune responses, certain B cell subsets may play negative immunomodulatory roles in different autoimmune diseases, which could be related to the secretion of cytokines and the initiation of inflammatory responses9. Additionally, there is growing support for the role of T cells in the progression of NMOSD10,11. IgG1 is the predominant subclass of AQP4-specific antibodies (AQP4-abs) in NMOSD patients and represents a T cell-dependent IgG subtype12. But AQP4-abs alone do not trigger inflammatory responses in the CNS13. When AQP4-specific T cells are co-transplanted with AQP4-IgG into rats, it results in tissue inflammation similar to that seen in NMOSD14, indicating the critical involvement of these T cells in the progression of NMOSD. Furthermore, AQP4-specific T cells have been detected in NMOSD patients, particularly those with a Th17 cell profile15. Research also suggests that the T cell receptor (TCR) could serve as a biomarker for diagnosing and treating autoimmune disorders and malignancies16,17. However, there have been insufficient studies on TCR characteristics and their value in diagnosis and prognosis. Importantly, while current therapies targeting B cells (e.g., rituximab) show benefit, a significant proportion of patients still experience relapse, highlighting the need for a deeper understanding of the broader immune dysregulation, particularly involving T cells, to identify novel therapeutic targets and biomarkers for improved treatment strategies. In this study, we performed an in-depth analysis of lymphocyte subsets in blood samples from individuals with NMOSD and healthy controls. Our goal was to characterize peripheral lymphocyte profiles in NMOSD patients, providing insights into immune dysregulation patterns that may inform future mechanistic studies Exploring the expression and function of B lymphocyte subsets in CNS inflammatory demyelinating diseases is important for understanding NMOSD. Therefore, this research sought to assess the levels of lymphocyte subsets in NMOSD patients to enhance our comprehension of the immune processes and disease mechanisms underlying NMOSD. Subjects and Methods 2.1 Subjects A total of 22 NMOSD patients treated in the Department of Neurology of the Affiliated Wuxi People’s Hospital of Nanjing Medical University between January 2018 and June 2023 comprised the NMOSD group, which included 21 female and one male patients. The mean age was 39.09 ± 17.24 years (mean ± standard deviation, SD), and the disease duration ranged from 1 to 33 years (defined as time from first symptom to sample collection). The diagnosis followed China's 2021 NMOSD Guidelines 18 {18} and jointly confirmed by two senior attending physicians, with serum AQP4-IgG detected by indirect immunofluorescence technique (tissue-based assay + cell-based assay ) . Peripheral blood samples were collected at initial admission prior to any immunosuppressive treatment for the current relapse. Disease duration was defined as time from first symptom to blood sampling. Exclusion criteria included: Patients with other autoimmune diseases, malignancies, acute infection, organ transplantation or HIV infection; Use of immunoglobulin, high-dose glucocorticoids or immunomodulatory drugs (such as mycophenolate, rituximab, azathioprine) within 3 months prior to sampling. Prior B cell depleting therapies (e.g., rituximab) within 12 months were also excluded. The control group comprised 22 age-matched individuals without autoimmune disease, and no recent infections or immunosuppressant use, including 13 females and 9 males. The mean age was (54.18 ± 20.19) years.The research received ethical approval from the Ethics Committee of the Wuxi People’s Hospital affiliated to Nanjing Medical University. All research was performed in accordance with relevant guidelines/regulations. Informed consent was obtained from all participants. Research was performed in accordance with the Declaration of Helsinki. 2.2 Study Methods Flow cytometry was performed to quantify lymphocyte subsets: T cells (CD3+), helper T cells (CD3 + CD4+), cytotoxic T cells (CD3 + CD8+), B cells (CD19+), and NK cells (CD3-CD16/56+).\" In the morning after admission, venous blood was collected from the antecubital vein for lymphocyte subsets and routine blood tests (including Red Blood Cell Count (RBC), Hemoglobin (Hb), Hematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), White Blood Cell Count (WBC), absolute and percentage counts of neutrophils, lymphocytes, monocytes, eosinophils, and basophils, and Platelet Count (PLT)). The percentages (CD3 + cells, CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD19 + B cells, CD3-CD(16/56) + NK cells, CD4+/CD8 + ratio) and the lymphocyte percentage from the routine blood tests in peripheral blood were collected from NMOSD and control group. Data analysis was performed using the SPSS 22.0 software. An independent samples t-test was employed to compare the two groups, with a P-value less than 0.05 indicating statistical significance. Pearson’s correlation coefficient, was used to evaluate the relationship between the duration of the disease and the lymphocyte subsets. Results The analysis of lymphocyte subsets between the two groups revealed that the percentage of CD3 + CD8 + T cells in the NMOSD patient group was higher compared to the control group. Conversely, the CD4+/CD8 + ratio and the lymphocyte percentage from routine blood tests in the NMOSD group were lower than those in the control group, with these differences being statistically significant (p < 0.05), as detailed in Table 1 . No significant intergroup differences were detected in: (a) CD3 + T cell populations (total CD3 + T cells and CD3 + CD4 + helper T cells], (b) CD19 + B cells, or (c) CD3-CD(16/56) + natural killer (NK) cells (all p > 0.05; Table 2 ). The percentages of CD3 + cells, CD3 + CD4 + T cells, CD3 + CD8 + T cells, CD19 + B cells, CD3-CD(16/56) + NK cells, CD4+/CD8 + ratio, and the peripheral blood lymphocyte percentage for both groups are depicted in Fig. 1 . The lymphocyte percentage was found to have a positive correlation with the duration of the disease (r² = 0.2529, p = 0.017, Pearson's correlation) as shown in Fig. 2 . Table 1 Comparison of the percentages of CD3 + CD8 + Tcell、CD4+/CD8 + and lymphocyte percentage in blood routine tests between the two groups Parameter Control group (n = 22) NMOSD group (n = 22) t-value p-value Flow cytometry subsets CD3 + CD8 + T cell% 22.86 ± 10.97 30.42 ± 9.55 2.437 0.019* CD4+/CD8 + ratio% 1.94 ± 0.85 1.28 ± 0.57 -3.031 0.004** Routine blood test Lymphocyte percentage, % 23.90 ± 12.78 16.52 ± 11.22 -2.034 0.048* Data presented as mean ± SD. Statistical analysis by independent samples t-test. *: A difference was considered statistically significant at p < 0.05. Table 2 Comparison of the percentages of CD3 + cells, CD3 + CD4 + T cells, CD19 + B cells, and CD3- CD(16/56) + NK cells between the two groups Parameter Control group (n = 22) NMOSD group (n = 22) t-value p-value Flow cytometry subsets CD3 + cell% 65.44 ± 14.20 68.55 ± 11.16 0.809 0.423 CD3 + CD4 + Tcell% 38.32 ± 11.40 34.99 ± 8.24 -1.108 0.274 CD19 + B cell% 18.58 ± 9.71 17.95 ± 9.16 -2.034 0.824 CD3- CD(16/56) + NK cell% 12.61 ± 6.55 10.58 ± 8.33 -0.900 0.373 Data presented as mean ± SD. Statistical analysis by independent samples t-test. Discussion Our study revealed a significant reduction in peripheral lymphocyte percentage and a reduced CD4+/CD8 + ratio in NMOSD patients compared to controls, which suggests that lymphocyte dysregulation may be implicated in NMOSD pathogenesis. This finding of lower lymphocyte percentage, and this measure showed a positive correlation with disease duration. This suggests that irregularities in lymphocytes levels might be linked to the development of NMOSD. Given that NMOSD is recognized as an autoimmune disorder with T lymphocytes playing a key role in immunopathology 19 , 20 , we further investigated T cell subsets. Crucially, we observed an elevated proportion of CD3 + CD8 + T cells in NMOSD patients compared to healthy controls. This increase in cytotoxic T cells suggests their potential contribution to NMOSD progression. The finding is consistent with previous studies demonstrating the importance of T cell clonal expansion in NMOSD development 21 . Furthermore, previous studies have shown that C perfringens peptide cross-reacts with the AQP4 antigen peptide to stimulate the activity of autoreactive T cells 22 , potentially contributing to the CD8 + T cell involvement we observed. Additionally, our data showed a significantly reduced CD4+/CD8 + T cell ratio in NMOSD patients, a pattern also reported in contexts like CMV infection. In NMOSD patients who are AQP4-IgG positive, there is a higher proportion of neutrophils and effector CD8 + T cells compared to healthy individuals 10 , 23 , 24 . Critically, the prominence of CD8 + T cells in our peripheral findings is strongly supported by histopathological evidence. Post-mortem examinations of NMOSD patients' spinal cords have shown extensive infiltration of CD8 + T cells in the subcortical white matter, optic chiasm, brainstem, and spinal cord, accompanied by macrophage activation 25 . Activated CD8 + T cells play a key role in disease initiation. Another case study highlighted the characteristic infiltration of T cells and macrophages in the leptomeninges and optic nerve in NMOSD 26 . Earlier studies have also identified T cells in the affected tissues of NMOSD patients and detected AQP4-specific T cells in their peripheral blood 22 , 27 . In the case of NMOSD, the inflammatory response triggered by T cells is part of the disease process 28 . Inflammatory damage of NMOSD leads to astrocytic lesions, characterized by various astrocytic pathologies, which are the pathological basis of CNS damage and dysfunction 29 . The lesions in astrocytes caused by activated cytotoxic T cells might be a result of an autoimmune reaction or a response to unidentified pathogens. Upon local activation, these T cells foster an inflammatory environment within the central nervous system (CNS), which can lead to NMOSD-like damage in astrocytes through AQP4 autoantibodies 30 . The precise function of T cells in the development of such astrocytic damage remains unclear. Research indicates that the mere presence of AQP4-specific autoantibodies in the CNS, even under conditions of inflammation and compromised blood-brain barrier, is insufficient to induce NMOSD-related astrocyte damage, and must be activated by additional effector mechanisms, such as complement-mediated and antibody-dependent cytotoxicity. When T cells inside the CNS are activated by specific antigens, the inflammatory response triggered by T cells constitutes this effector environment. This mechanism may be similar to the occurrence of human NMOSD lesions because invading T cells show signs of activation 30 . Our findings of peripheral CD8 + T -cell expansion and altered CD4/CD8 ratio add further weight to the significance of T cell dysregulation, particularly cytotoxic effector functions, in the peripheral immunobiology and potentially the CNS pathogenesis of NMOSD. This study has several limitations worthy of consideration. Firstly, our flow cytometry analysis focused on major T cell subsets (CD4+, CD8+), without characterizing more refined phenotypic or functional subsets (such as Th17, Treg, memory subsets, activation markers), which may provide deeper mechanism insights into NMOSD immunopathology. Secondly, the analysis was confined to peripheral blood, correlating these findings with lymphocyte profiles and activation states within the cerebrospinal fluid (CSF) or directly within CNS lesions would offer a more comprehensive view of the disease process. Thirdly, the study design was cross-sectional, which limited our ability to infer causal relationships between the observed lymphocyte alterations and the occurrence or progression of the disease. Finally, when we observe correlations, the sample size may limit the ability to detect more subtle associations or fully explain potential confounding factors. Conclusion Our findings suggest that the dynamic balance of the immune system is critical for maintaining its stability, but the specific pathogenesis of NMOSD remains incompletely understood. Our research indicated notable distinctions between individuals with NMOSD and healthy controls, particularly regarding elevated percentages of CD3 + CD8 + T cells, a reduced CD4+/CD8 + ratio, and a lower lymphocyte percentage found in peripheral blood. The lymphocyte percentage showed a modest positive correlation with the duration of the disease, hinting at the potential significance of these immunological markers in the advancement of NMOSD. Although current therapeutic approaches for neuromyelitis optica spectrum disorder (NMOSD) and other immune disorders increasingly focus on B cells, our data indicate that exploring CD3 + CD8 + T cells as additional potential immunotherapy targets may be justified. Continuous monitoring and retrospective analysis could facilitate the development of novel diagnostic and treatment strategies for NMOSD. Moreover, this investigation was confined to a single institution, where the incidence of NMOSD was relatively low, leading to a limited sample size. Our findings emphasize CD3 + CD8 + T-cell expansion and altered CD4/CD8 ratios as characteristics of NMOSD immunopathology, thus necessitating validation in larger longitudinal studies. Consequently, the findings, especially the correlation analysis, should be interpreted with caution. In the future, it is anticipated that multicenter, large-scale sample, longitudinal, or prospective randomized controlled trials will be conducted to verify and enrich the findings of this study, confirm these immunological associations, and explore their clinical significance. Declarations Ethics approval and consent to participate The research have been performed in accordance with the Declaration of Helsinki and have been approved by the Ethics Committee of the Wuxi People’s Hospital affiliated to Nanjing Medical University. Informed consent was obtained from all participants. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests\" in this section. Funding Not applicable Authors' contributions Shi made all the contributions to the research and carry all the responsibilities. Acknowledgements Not applicable References Levin MH, Bennett JL, Verkman AS. Optic neuritis in neuromyelitis optica. Prog Retin Eye Res. 2013;36:159–71. 10.1016/j.preteyeres.2013.03.001 . Wu Y, Zhong L, Geng J. Neuromyelitis optica spectrum disorder: Pathogenesis, treatment, and experimental models. Mult Scler Relat Disord. 2019;27:412–8. 10.1016/j.msard.2018.12.002 . Shi Z, et al. Effects of immunotherapies and prognostic predictors in neuromyelitis optica spectrum disorder: a prospective cohort study. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7291609\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":511782048,\"identity\":\"3de192a7-b6a5-446d-ad86-6416fe43f55d\",\"order_by\":0,\"name\":\"Shi Qi\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie3RMQrCMBSA4VcCweFJNwko5ApPXDoIXiVddHHoERTBSfeWXqJHiGT1ABUFlV4gYwdBq7gpNqND/iFk+UjyAuDz/WMiWICiO07Olda2diYJG4Cexrts40Sei2Vj0POR6XAHIfPVsrwQx2CxtwYQZNjTvwmddqtIESILtoVJIhhmuWohIl4LRQI56xYmxWYUxxYi0xdpjuF4McgdCJQvolAgghuhMn6+RSMJTs2QRftbZDq7HuqbnpBglbX1WIb9tovB+2u+7B2Jz+fz+T57ACkuQ+jI/o49AAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"the Affiliated Wuxi People’s Hospital of Nanjing Medical University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Shi\",\"middleName\":\"\",\"lastName\":\"Qi\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-08-04 13:23:14\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7291609/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7291609/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":90981635,\"identity\":\"352eee17-e9d6-4a66-9001-0cc15d80b006\",\"added_by\":\"auto\",\"created_at\":\"2025-09-10 09:25:08\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":173788,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePercentages of CD3+ cell, CD3+ CD4+ T cell, CD3+ CD8+ T cell, CD19+ B cell, CD3-CD (16/56) + NK cell, CD4+/CD8+ ratio and lymphocyte percentage in routine blood tests between the two groups.\\u003c/p\\u003e\\n\\u003cp\\u003e*: A difference was considered statistically significant at p\\u0026lt;0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7291609/v1/6a06df364f8b560698cff353.jpg\"},{\"id\":90981636,\"identity\":\"87b21f00-640f-4a16-951f-8d630d42ae7f\",\"added_by\":\"auto\",\"created_at\":\"2025-09-10 09:25:08\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":92590,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCorrelation between lymphocyte percentage and disease duration\\u003c/p\\u003e\\n\\u003cp\\u003eNMOSD: neuromyelitis spectrum disorder\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Picture2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7291609/v1/fefd283b2cd8935554a6a8d5.jpg\"},{\"id\":91474864,\"identity\":\"a56a906d-38c7-4512-930e-5e2f92a0ea7a\",\"added_by\":\"auto\",\"created_at\":\"2025-09-17 00:16:33\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":649537,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7291609/v1/6fea4a00-c7f6-43bd-935e-ac59d859ad98.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Characteristics of peripheral blood lymphocyte subsets in patients with neuromyelitis optica\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eDevic\\u0026rsquo;s disease, also known as neuromyelitis spectrum disorder (NMOSD), is a rare inflammatory condition affecting the central nervous system (CNS). It is marked by an autoimmune attack primarily targeting the optic nerve and/or the spinal cord. Population and epidemiological studies indicate that the incidence of NMOSD is relatively low, at approximately 0.3 to 4.4 cases per 100,000 people. NMOSD accounts for a higher proportion among Asian patients with demyelinating disease, ranging between 20% and 48%1. Most individuals with NMOSD are aged 35 to 45 age bracket, though the condition can also affect both children and older adults. With the identification of the AQP4-IgG antibody, which targets aquaporin-4, a key water channel in the CNS, NMOSD is increasingly recognized as a distinct disorder from multiple sclerosis2. It has a high recurrence rate and leads to high rates of disability. Prognostic studies indicate that without intervention, patients with NMOSD face a 58\\u0026ndash;83% chance of developing motor dysfunction within five years3,4. Current management strategies for NMOSD primarily involve the use of conventional immunosuppressive and immunomodulatory medications. However, more than 30% of NMOSD patients are at risk of recurrence and deterioration. The pathological mechanism of NMOSD is complex, but recently, there have been advances in treatment options, such as eculizumab, satralizumab, etc. In the acute stage, treatments typically involve hormones and plasma exchange, whereas long-term immunosuppressants, particularly those that target B cells, are employed to prevent relapse, which significantly improves the long-term quality of life of patients5. There are several hypotheses on the immunopathogenesis of NMOSD, including the generation of anti-AQP4 antibodies, elevated activity of inflammatory B cell and plasmablasts, dysfunctional B cell tolerance checkpoints, reduced regulatory B cell activity, and the disruption of B cell anergy6. B cells are crucial in the pathogenesis of NMOSD, exerting their influence through various mechanisms. Initially, B cells differentiate into plasmablasts and plasma cells, which produce the disease-causing AQP4-IgG7. Additionally, B cell release inflammatory cytokines, such as IL-6, which can trigger both harmful and inflammatory immune responses. Furthermore, B cells can function as antigen-presenting cells, facilitating the maturation and activation of autoreactive T cells6,7. Lastly, the depletion and subsequent restoration of B cells aids in the recovery of the number and functionality of regulatory B cells8. Recent studies have shown that in addition to B lymphocyte-mediated humoral immune responses, certain B cell subsets may play negative immunomodulatory roles in different autoimmune diseases, which could be related to the secretion of cytokines and the initiation of inflammatory responses9. Additionally, there is growing support for the role of T cells in the progression of NMOSD10,11. IgG1 is the predominant subclass of AQP4-specific antibodies (AQP4-abs) in NMOSD patients and represents a T cell-dependent IgG subtype12. But AQP4-abs alone do not trigger inflammatory responses in the CNS13. When AQP4-specific T cells are co-transplanted with AQP4-IgG into rats, it results in tissue inflammation similar to that seen in NMOSD14, indicating the critical involvement of these T cells in the progression of NMOSD. Furthermore, AQP4-specific T cells have been detected in NMOSD patients, particularly those with a Th17 cell profile15. Research also suggests that the T cell receptor (TCR) could serve as a biomarker for diagnosing and treating autoimmune disorders and malignancies16,17. However, there have been insufficient studies on TCR characteristics and their value in diagnosis and prognosis. Importantly, while current therapies targeting B cells (e.g., rituximab) show benefit, a significant proportion of patients still experience relapse, highlighting the need for a deeper understanding of the broader immune dysregulation, particularly involving T cells, to identify novel therapeutic targets and biomarkers for improved treatment strategies. In this study, we performed an in-depth analysis of lymphocyte subsets in blood samples from individuals with NMOSD and healthy controls. Our goal was to characterize peripheral lymphocyte profiles in NMOSD patients, providing insights into immune dysregulation patterns that may inform future mechanistic studies Exploring the expression and function of B lymphocyte subsets in CNS inflammatory demyelinating diseases is important for understanding NMOSD. Therefore, this research sought to assess the levels of lymphocyte subsets in NMOSD patients to enhance our comprehension of the immune processes and disease mechanisms underlying NMOSD.\\u003c/p\\u003e\"},{\"header\":\"Subjects and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec2\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.1 Subjects\\u003c/h2\\u003e\\u003cp\\u003eA total of 22 NMOSD patients treated in the Department of Neurology of the Affiliated Wuxi People\\u0026rsquo;s Hospital of Nanjing Medical University between January 2018 and June 2023 comprised the NMOSD group, which included 21 female and one male patients. The mean age was 39.09\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;17.24 years (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation, SD), and the disease duration ranged from 1 to 33 years (defined as time from first symptom to sample collection). The diagnosis followed China's 2021 NMOSD Guidelines\\u003csup\\u003e\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e{18} and jointly confirmed by two senior attending physicians, with serum AQP4-IgG detected by indirect immunofluorescence technique (tissue-based assay\\u0026thinsp;+\\u0026thinsp;cell-based assay\\u003cb\\u003e)\\u003c/b\\u003e. Peripheral blood samples were collected at initial admission prior to any immunosuppressive treatment for the current relapse. Disease duration was defined as time from first symptom to blood sampling. Exclusion criteria included: Patients with other autoimmune diseases, malignancies, acute infection, organ transplantation or HIV infection; Use of immunoglobulin, high-dose glucocorticoids or immunomodulatory drugs (such as mycophenolate, rituximab, azathioprine) within 3 months prior to sampling. Prior B cell depleting therapies (e.g., rituximab) within 12 months were also excluded.\\u003c/p\\u003e\\u003cp\\u003eThe control group comprised 22 age-matched individuals without autoimmune disease, and no recent infections or immunosuppressant use, including 13 females and 9 males. The mean age was (54.18\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;20.19) years.The research received ethical approval from the Ethics Committee of the Wuxi People\\u0026rsquo;s Hospital affiliated to Nanjing Medical University. All research was performed in accordance with relevant guidelines/regulations. Informed consent was obtained from all participants. Research was performed in accordance with the Declaration of Helsinki.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.2 Study Methods\\u003c/h2\\u003e\\u003cp\\u003eFlow cytometry was performed to quantify lymphocyte subsets: T cells (CD3+), helper T cells (CD3\\u0026thinsp;+\\u0026thinsp;CD4+), cytotoxic T cells (CD3\\u0026thinsp;+\\u0026thinsp;CD8+), B cells (CD19+), and NK cells (CD3-CD16/56+).\\\"\\u003c/p\\u003e\\u003cp\\u003eIn the morning after admission, venous blood was collected from the antecubital vein for lymphocyte subsets and routine blood tests (including Red Blood Cell Count (RBC), Hemoglobin (Hb), Hematocrit (Hct), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Red Cell Distribution Width (RDW), White Blood Cell Count (WBC), absolute and percentage counts of neutrophils, lymphocytes, monocytes, eosinophils, and basophils, and Platelet Count (PLT)). The percentages (CD3\\u0026thinsp;+\\u0026thinsp;cells, CD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;T cells, CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells, CD19\\u0026thinsp;+\\u0026thinsp;B cells, CD3-CD(16/56)\\u0026thinsp;+\\u0026thinsp;NK cells, CD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio) and the lymphocyte percentage from the routine blood tests in peripheral blood were collected from NMOSD and control group. Data analysis was performed using the SPSS 22.0 software. An independent samples t-test was employed to compare the two groups, with a P-value less than 0.05 indicating statistical significance. Pearson\\u0026rsquo;s correlation coefficient, was used to evaluate the relationship between the duration of the disease and the lymphocyte subsets.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eThe analysis of lymphocyte subsets between the two groups revealed that the percentage of CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells in the NMOSD patient group was higher compared to the control group. Conversely, the CD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio and the lymphocyte percentage from routine blood tests in the NMOSD group were lower than those in the control group, with these differences being statistically significant (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), as detailed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. No significant intergroup differences were detected in: (a) CD3\\u0026thinsp;+\\u0026thinsp;T cell populations (total CD3\\u0026thinsp;+\\u0026thinsp;T cells and CD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;helper T cells], (b) CD19\\u0026thinsp;+\\u0026thinsp;B cells, or (c) CD3-CD(16/56)\\u0026thinsp;+\\u0026thinsp;natural killer (NK) cells (all p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05; Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). The percentages of CD3\\u0026thinsp;+\\u0026thinsp;cells, CD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;T cells, CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells, CD19\\u0026thinsp;+\\u0026thinsp;B cells, CD3-CD(16/56)\\u0026thinsp;+\\u0026thinsp;NK cells, CD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio, and the peripheral blood lymphocyte percentage for both groups are depicted in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. The lymphocyte percentage was found to have a positive correlation with the duration of the disease (r\\u0026sup2; = 0.2529, p\\u0026thinsp;=\\u0026thinsp;0.017, Pearson's correlation) as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eComparison of the percentages of CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;Tcell、CD4+/CD8\\u0026thinsp;+\\u0026thinsp;and lymphocyte percentage in blood routine tests between the two groups\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eParameter\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eControl group (n\\u0026thinsp;=\\u0026thinsp;22)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNMOSD group (n\\u0026thinsp;=\\u0026thinsp;22)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003et-value\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003ep-value\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eFlow cytometry subsets\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cell%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e22.86\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;10.97\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e30.42\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9.55\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e2.437\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.019*\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e1.94\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.85\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e1.28\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.57\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-3.031\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.004**\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eRoutine blood test\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eLymphocyte percentage, %\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e23.90\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;12.78\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e16.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.22\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-2.034\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.048*\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eData presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD.\\u003c/p\\u003e\\u003cp\\u003eStatistical analysis by independent samples t-test.\\u003c/p\\u003e\\u003cp\\u003e*: A difference was considered statistically significant at p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eComparison of the percentages of CD3\\u0026thinsp;+\\u0026thinsp;cells, CD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;T cells, CD19\\u0026thinsp;+\\u0026thinsp;B cells, and CD3- CD(16/56)\\u0026thinsp;+\\u0026thinsp;NK cells between the two groups\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eParameter\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eControl group (n\\u0026thinsp;=\\u0026thinsp;22)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNMOSD group (n\\u0026thinsp;=\\u0026thinsp;22)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003et-value\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003ep-value\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eFlow cytometry subsets\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCD3\\u0026thinsp;+\\u0026thinsp;cell%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e65.44\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;14.20\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e68.55\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.16\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e0.809\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.423\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;Tcell%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e38.32\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;11.40\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e34.99\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;8.24\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-1.108\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.274\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCD19\\u0026thinsp;+\\u0026thinsp;B cell%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e18.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9.71\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e17.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;9.16\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-2.034\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.824\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eCD3- CD(16/56)\\u0026thinsp;+\\u0026thinsp;NK cell%\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e12.61\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;6.55\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\"\\u0026plusmn;\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003e10.58\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;8.33\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003e-0.900\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003e0.373\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eData presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD.\\u003c/p\\u003e\\u003cp\\u003eStatistical analysis by independent samples t-test.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eOur study revealed a significant reduction in peripheral lymphocyte percentage and a reduced CD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio in NMOSD patients compared to controls, which suggests that lymphocyte dysregulation may be implicated in NMOSD pathogenesis. This finding of lower lymphocyte percentage, and this measure showed a positive correlation with disease duration. This suggests that irregularities in lymphocytes levels might be linked to the development of NMOSD. Given that NMOSD is recognized as an autoimmune disorder with T lymphocytes playing a key role in immunopathology\\u003csup\\u003e\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e, we further investigated T cell subsets. Crucially, we observed an elevated proportion of CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells in NMOSD patients compared to healthy controls. This increase in cytotoxic T cells suggests their potential contribution to NMOSD progression. The finding is consistent with previous studies demonstrating the importance of T cell clonal expansion in NMOSD development\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. Furthermore, previous studies have shown that C perfringens peptide cross-reacts with the AQP4 antigen peptide to stimulate the activity of autoreactive T cells\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u003c/sup\\u003e, potentially contributing to the CD8\\u0026thinsp;+\\u0026thinsp;T cell involvement we observed. Additionally, our data showed a significantly reduced CD4+/CD8\\u0026thinsp;+\\u0026thinsp;T cell ratio in NMOSD patients, a pattern also reported in contexts like CMV infection. In NMOSD patients who are AQP4-IgG positive, there is a higher proportion of neutrophils and effector CD8\\u0026thinsp;+\\u0026thinsp;T cells compared to healthy individuals\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u003c/sup\\u003e. Critically, the prominence of CD8\\u0026thinsp;+\\u0026thinsp;T cells in our peripheral findings is strongly supported by histopathological evidence. Post-mortem examinations of NMOSD patients' spinal cords have shown extensive infiltration of CD8\\u0026thinsp;+\\u0026thinsp;T cells in the subcortical white matter, optic chiasm, brainstem, and spinal cord, accompanied by macrophage activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. Activated CD8\\u0026thinsp;+\\u0026thinsp;T cells play a key role in disease initiation. Another case study highlighted the characteristic infiltration of T cells and macrophages in the leptomeninges and optic nerve in NMOSD\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. Earlier studies have also identified T cells in the affected tissues of NMOSD patients and detected AQP4-specific T cells in their peripheral blood\\u003csup\\u003e\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. In the case of NMOSD, the inflammatory response triggered by T cells is part of the disease process\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. Inflammatory damage of NMOSD leads to astrocytic lesions, characterized by various astrocytic pathologies, which are the pathological basis of CNS damage and dysfunction\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e. The lesions in astrocytes caused by activated cytotoxic T cells might be a result of an autoimmune reaction or a response to unidentified pathogens. Upon local activation, these T cells foster an inflammatory environment within the central nervous system (CNS), which can lead to NMOSD-like damage in astrocytes through AQP4 autoantibodies\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. The precise function of T cells in the development of such astrocytic damage remains unclear. Research indicates that the mere presence of AQP4-specific autoantibodies in the CNS, even under conditions of inflammation and compromised blood-brain barrier, is insufficient to induce NMOSD-related astrocyte damage, and must be activated by additional effector mechanisms, such as complement-mediated and antibody-dependent cytotoxicity. When T cells inside the CNS are activated by specific antigens, the inflammatory response triggered by T cells constitutes this effector environment. This mechanism may be similar to the occurrence of human NMOSD lesions because invading T cells show signs of activation\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Our findings of peripheral CD8\\u0026thinsp;+\\u0026thinsp;T -cell expansion and altered CD4/CD8 ratio add further weight to the significance of T cell dysregulation, particularly cytotoxic effector functions, in the peripheral immunobiology and potentially the CNS pathogenesis of NMOSD.\\u003c/p\\u003e\\u003cp\\u003eThis study has several limitations worthy of consideration. Firstly, our flow cytometry analysis focused on major T cell subsets (CD4+, CD8+), without characterizing more refined phenotypic or functional subsets (such as Th17, Treg, memory subsets, activation markers), which may provide deeper mechanism insights into NMOSD immunopathology. Secondly, the analysis was confined to peripheral blood, correlating these findings with lymphocyte profiles and activation states within the cerebrospinal fluid (CSF) or directly within CNS lesions would offer a more comprehensive view of the disease process. Thirdly, the study design was cross-sectional, which limited our ability to infer causal relationships between the observed lymphocyte alterations and the occurrence or progression of the disease. Finally, when we observe correlations, the sample size may limit the ability to detect more subtle associations or fully explain potential confounding factors.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eOur findings suggest that the dynamic balance of the immune system is critical for maintaining its stability, but the specific pathogenesis of NMOSD remains incompletely understood. Our research indicated notable distinctions between individuals with NMOSD and healthy controls, particularly regarding elevated percentages of CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells, a reduced CD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio, and a lower lymphocyte percentage found in peripheral blood. The lymphocyte percentage showed a modest positive correlation with the duration of the disease, hinting at the potential significance of these immunological markers in the advancement of NMOSD. Although current therapeutic approaches for neuromyelitis optica spectrum disorder (NMOSD) and other immune disorders increasingly focus on B cells, our data indicate that exploring CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells as additional potential immunotherapy targets may be justified. Continuous monitoring and retrospective analysis could facilitate the development of novel diagnostic and treatment strategies for NMOSD.\\u003c/p\\u003e\\u003cp\\u003eMoreover, this investigation was confined to a single institution, where the incidence of NMOSD was relatively low, leading to a limited sample size. Our findings emphasize CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T-cell expansion and altered CD4/CD8 ratios as characteristics of NMOSD immunopathology, thus necessitating validation in larger longitudinal studies.\\u003c/p\\u003e\\u003cp\\u003eConsequently, the findings, especially the correlation analysis, should be interpreted with caution. In the future, it is anticipated that multicenter, large-scale sample, longitudinal, or prospective randomized controlled trials will be conducted to verify and enrich the findings of this study, confirm these immunological associations, and explore their clinical significance.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eEthics approval and consent to participate\\u003c/p\\u003e\\n\\u003cp\\u003eThe research have been performed in accordance with the Declaration of Helsinki and have been approved by the Ethics Committee of the Wuxi People\\u0026rsquo;s Hospital affiliated to Nanjing Medical University. Informed consent was obtained from all participants.\\u003c/p\\u003e\\n\\u003cp\\u003eConsent for publication\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003eAvailability of data and materials\\u003c/p\\u003e\\n\\u003cp\\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003eCompeting interests\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests\\u0026quot; in this section.\\u003c/p\\u003e\\n\\u003cp\\u003eFunding\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003eAuthors\\u0026apos; contributions\\u003c/p\\u003e\\n\\u003cp\\u003eShi made all the contributions to the research and carry all the responsibilities.\\u003c/p\\u003e\\n\\u003cp\\u003eAcknowledgements\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eLevin MH, Bennett JL, Verkman AS. 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Mult Scler. 2010;16:1443\\u0026ndash;52. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1177/1352458510379247\\u003c/span\\u003e\\u003cspan address=\\\"10.1177/1352458510379247\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eLucchinetti CF, et al. The pathology of an autoimmune astrocytopathy: lessons learned from neuromyelitis optica. Brain Pathol. 2014;24:83\\u0026ndash;97. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1111/bpa.12099\\u003c/span\\u003e\\u003cspan address=\\\"10.1111/bpa.12099\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003ePohl M, et al. T cell-activation in neuromyelitis optica lesions plays a role in their formation. Acta Neuropathol Commun. 2013;1:85. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003e10.1186/2051-5960-1-85\\u003c/span\\u003e\\u003cspan address=\\\"10.1186/2051-5960-1-85\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"neuromyelitis optica (NMO), NMO spectrum disorder (NMOSD), autoimmune inflammation, aquaporin-4 immunoglobulin G antibody (AQP4-IgG), lymphocyte subsets\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7291609/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7291609/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBackground: Neuromyelitis spectrum disorder (NMOSD) is inflammatory disease of the central nervous system (CNS), mainly affecting the optic nerve and/or spinal cord, and characterized by the presence of aquaporin-4 immunoglobulin G antibody (AQP4-IgG) in the serum. The role and activity of different lymphocyte subsets are crucial for understanding the immune mechanisms and pathogenesis of NMOSD. This study aimed to analyze the levels of lymphocyte subsets in NMOSD patients.\\u003c/p\\u003e\\u003cp\\u003eMethods: 22 NMOSD patients and 22 individuals without autoimmune diseases were enrolled. Lymphocyte subsets were measured in both groups, including CD3\\u0026thinsp;+\\u0026thinsp;cells, CD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;T cells, CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells, CD19\\u0026thinsp;+\\u0026thinsp;B cells, CD3- CD(16/56)\\u0026thinsp;+\\u0026thinsp;natural killer cells (NK cells), and CD4+/CD8\\u0026thinsp;+\\u0026thinsp;ratio, along with the routine peripheral blood lymphocyte percentage. The data were analyzed using SPSS 22.0.\\u003c/p\\u003e\\u003cp\\u003eResults: The total lymphocyte percentage in routine peripheral blood tests were significantly lower in the NMOSD group compared to controls (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05). Lymphocyte subset analysis revealed the NMOSD group had a significantly higher proportion of CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells compared to controls. Additionally, the CD4+/CD8\\u0026thinsp;+\\u0026thinsp;T cell ratio was significantly lower in the NMOSD group. No significant differences were observed in the proportions of CD3\\u0026thinsp;+\\u0026thinsp;cells, CD3\\u0026thinsp;+\\u0026thinsp;CD4\\u0026thinsp;+\\u0026thinsp;T cells, CD19\\u0026thinsp;+\\u0026thinsp;B cells, or CD3-CD56/16\\u0026thinsp;+\\u0026thinsp;NK cells between groups. The total lymphocyte percentage showed a positively correlated with the disease duration(r\\u0026sup2; = 0.2529, P\\u0026thinsp;=\\u0026thinsp;0.017).\\u003c/p\\u003e\\u003cp\\u003eConclusion: This study suggests that T cell levels, and CD4/CD8 ratio are connected with the occurrence and development of NMOSD. We observed a reduced total lymphocyte percentage in routine blood tests, an elevated proportion of CD3\\u0026thinsp;+\\u0026thinsp;CD8\\u0026thinsp;+\\u0026thinsp;T cells and a decreased CD4+/CD8\\u0026thinsp;+\\u0026thinsp;T cell ratio compared to controls. These immunological changes show associations with NMOSD status, with the total lymphocyte percentage positively correlating with disease duration. Further investigation of these immune parameters may offer potential avenues for monitoring disease progression and developing adjunctive therapeutic strategies for NMOSD.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Characteristics of peripheral blood lymphocyte subsets in patients with neuromyelitis optica\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-09-10 09:25:03\",\"doi\":\"10.21203/rs.3.rs-7291609/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"a2d00f52-2e87-43f3-98f8-225e44985c43\",\"owner\":[],\"postedDate\":\"September 10th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-09-17T00:08:19+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-09-10 09:25:03\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7291609\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7291609\",\"identity\":\"rs-7291609\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}