Preliminary Study On Follicular Helper T Cells In Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system(CNS) characterized by presence of inflammatory demyelinating lesions in white matter.The precise etiology and pathogenesis is still unclear. In conventional immunological view,cell immunity plays a key role in the occurrence of MS, but B cell-mediated humoralimmunity has attracted a increasing attention in recent years. The important role of B cellsand autoantibodies in the pathogenesis of MS has been supported by a large quantity ofevidence from animal experiments, autopsy, peripheral blood and cerebrospinal fluid (CSF)analysis, as well as B cell depletion therapy, however, it is not entirely clear how B cells isactivated and by which mechanism. Recent studies have indicated that follicular helper Tcells (Tfh), a T cell subset with phenotype of CD4+CXCR5+, can assist B cell activation,proliferation, differentiation and the formation of germinal center (GC). The discovery ofTfh cells (the true helper of B cell activation) is considered as a major breakthrough incollaborative research on T-B cells[1]. The changes in the function of CD4+CXCR5+Tcells under diseased situation has become one hot spot of revelant research. Studies inthe crosstalk of Tfh cells and B cells in experimental autoimmune encephalomyelitis (EAE), the classical animal model of MS is also needed.ObjectiveTo establish and evaluate EAE model in female C57BL/6mice; to observe theformation of germinal center in the spleens of EAE mice and the formation of ectopiclymph follicle-like structures in the spinal cords of EAE mice; to explore the changes inproporations of Tfh cells, B cells and their subgroups in peripheral immune organs andpathologic target organ during the course of EAE, and to provide the preliminary evidenceon the participation of Tfh cells in the pathogenesis of EAE.MethodsEAE was induced in female C57BL/6mice by immunization with myelinoligodendrocyte glycoprotein peptide35-55(MOG35-55), and pathological changes inspinal cord tissues were revealed by hematoxylin&eosin (HE) staining and Luxol fastblue (LFB) myelin staining. The formation of germinal center and the position changes ofdifferent immune cells in the spleens of EAE mice, as well as presence of ectopic lymphfollicle-like structures in spinal cords of EAE mice were determined by multipleimmunofluorescent staining. The proporations of Tfh cells, B cells and their subgroupsfrom spleens, lymphoid nodes and spinal cords at the different time points of EAE coursewere evaluated by flow cytometric analysis.ResultsMOG35-55peptide-immunized mice began to develop the EAE symptoms on day11postimmunization, and the incidence was mainly concentrated in the days12-16postimmunization. The peak of disease occurred after the onset of4.25±0.74days, withthe overall morbility of about90%. EAE mice presented with different degrees ofparalysis of limbs, especially of hind limbs. HE staining showed an extensive infiltrationof inflammatory cells in sections of spinal cords from EAE mice, mainly around bloodvessels and in the meninges. Luxol fast blue staining revealed multiple demyelinatedlesions in the above regions. By multiple immunofluorescent staining, the formation ofgerminal center in the spleens of EAE mice were revealed, accompanied by changes in thelocations of T cells, B cells, and follicular dendritic cells (FDC). Meanwhile, ectopic lymphoid follicle-like structures were discovered in the spinal cords of EAE mice. Also, alarge quantity of CD4+CXCR5+ICOS+Tfh cells and CD4+CXCR5+PD-1+Tfh cells wereobserved in the sections of spleens and spinal cords from EAE mice. Flow cytometricanalysis showed that the proporations of Tfh cells from the spleens, lymphoid nodes andspinal cords of EAE group on the pre-EAE, peak, remitting, and chronic persistent phaseswere significantly increased (P <0.05, compared with control group). Further cellsubgroup analysis revealed that the proporation of Th1-like Tfh cells was decreased, andthe proporations of Th2-like and Th17-like Tfh cells were increased. Similar with thetendency of Tfh cells, the proporations of different B cell subgroups from the aboveorgans of EAE mice were significantly higher than those from control mice at the differenttime points of EAE course.ConclusionEAE leads to the formation of germinal center in the spleens of EAE miceaccompanied by migration of different immune cells and a formation of ectopic lymphoidfollicle-like structures in the pathologic target organ, i.e. spinal cord. These findings,together with the persistent higher proporations of Tfh cells and B cell subgroups inperipheral immune organs and pathologic target organ during the course of EAE, provide apreliminary evidence on the participation of Tfh cells in pathogenesis of EAE.