IDENTIFICATION OF CELLULAR FACTORS ASSOCIATED WITH INFLAMMATION AND NEURODEGENERATION IN MULTIPLE SCLEROSIS

Alexander Rodero-Romero¹José Ignacio Fernández Velasco¹Enric Monreal²Raquel Sainz Amo²Roberto Alvarez-Lafuente³Manuel Comabella^4,5Lluís Ramió-Torrentà^6,7,8José M García-Dominguez⁹Noelia Villarrubia¹Susana Sainz de la Maza²María Domínguez-Mozo³Ana Quiroga-Varela⁶Juan Luís Chico-García²Fernando Rodriguez Jorge²José Luis Veiga-González¹Ernesto Roldan Santiago¹Mercedes Espiño¹Eulalia Rodríguez-Martín¹Gary Álvarez Bravo⁶Jaime MASJUAN²Xavier Montalban^4,5Lucienne Costa-Frossard²Luisa María Villar^1*

• ¹Department of Immunology, Hospital Universitario Ramón y Cajal, Red Española de Esclerosis Múltiple, Red de Enfermedades Inflamatorias, ISCIII, Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain
• ²Department of Neurology, Hospital Universitario Ramón y Cajal, Red Española de Esclerosis Múltiple, Red de Enfermedades Inflamatorias, ISCIII, Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain
• ³Grupo Investigación de Factores Ambientales en Enfermedades Degenerativas, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Madrid, Spain
• ⁴Servei de Neurologia, Centre d'Esclerosi Múltiple de Catalunya, Institut de Recerca Vall d'Hebron, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain
• ⁵Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED) - Instituto Sanitario Carlos III, Madrid, Spain
• ⁶Neurodegeneration and Neuroinflammation Research Group, Girona Biomedical Research Institute (IDIBGI-CERCA),, Salt, Spain
• ⁷Red de Enfermedades inflamatorias (RD24/0007/0005), Instituto de Salud Carlos III, Madrid, Spain
• ⁸Medical Sciences Department, Faculty of Medicine, Universitat de Girona, Girona, Spain
• ⁹Department of Neurology, Hospital General Universitario Gregorio Marañón, Madrid, Spain

Background: Serum biomarkers as neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) enabled early identification of multiple sclerosis (MS) patients at risk of relapse-associated worsening (RAW) or progression independent of relapses (PIRA). However, the immunological mechanisms underlying these clinical phenotypes remain unclear.Methods: We conducted a cross-sectional study including 117 MS patients and 84 healthy controls (HC). Patients were stratified as NLGL (low sNfL and sGFAP), NH (high sNfL at different levels of sGFAP), and NLGH (low sNfL and high sGFAP). Percentages of blood and cerebrospinal fluid (CSF) mononuclear cells, and intracellular production of cytokines by T and B cells after "in vitro" stimulation were analyzed by flow cytometry.Results: We identified a common inflammatory profile present in the blood of all MS groups comprising significant increases of effector CD4⁺ and CD8⁺ T cells, of memory and antigenpresenting B cells, of CD4 + and CD8 + T cells producing interferon-gamma, interleukin-17 and tumor necrosis factor-alpha (TNF-α) and of B cells producing TNF-α.Additionally, the highly inflammatory NH group showed lower frequencies of different regulatory subsets (transitional B cells, PDL1 + monocytes and Treg cells) compared to HC and increased percentages of CD4 + and CD8⁺ T cells producing granulocyte-macrophage colony-stimulating factor and of effector CD56 dim NK cells. They also showed lower percentages of Treg in blood and CSF compared to the low inflammatory NLGL group, which also displayed higher frequencies of regulatory CD56 dim , NKG2A⁺ cells. Conclusion: All MS patients share increased inflammatory B and T cells, but differ in regulatory or NK subsets, which identify highly inflammatory or benign disease courses.