Changes And Significance Of Circulating Memory T Follicular Helper Sub-group In Patients With Guillain-barré Syndrome

Background: Guillain Barre syndrome(GBS) is a worldwide distribution of acute immune mediated inflammatory disease that affects the peripheral nervous system(PNS). Despite its low incidence(0.001-0.002%) and self-limited course, there are still a few severe cases with visible axonal degeneration and fragmentation and carries 15%disability and 5% mortality. As reported, GBS consists of several subtypes: acute inflammatory demyelinating multiple radiculoneuropathy(AIDP), acute motor axonal neuropathy(AMAN), acute motor-sensory axonal neuropathy(AMSAN), Miller Fisher syndrome(MFS), acute panautonomic neuropathy, acute sensory neuropathy(ASN) and so on. The classification depends on an understanding of the involved nerve fiber types.The prototype of GBS, which accounts for 90% of all GBS cases in Europe and North America, is acute AIDP. AMAN and AMSAN are more prevalent in Asia, South and Central America. The current report highlights the differences in the immunological pathogenesis of various subtypes of GBS. AMAN, AMSAN and MFS are more related to autoantibodies against ganglioside, and anti-GQ1 b antibodies are present in approximately 95% of patients with MFS, while AIDP mainly involves CD4+ Th cell-induced macrophage- and complement-associated demyelination. A vast amount of study have indicated that the pathogenesis, clinical course, severity and outcome of different subtypes of GBS have a high degree of specificity. In 2014, the latest statistical analysis of clinical data from North China was reported by Zhang’s research team that ganglioside-associated GBS, which mainly involves axonal injury, is known to be associated with severe manifestations and poorer short-term prognosis. In 2015,Zhang G reported that the prognosis of AMAN group was poorer than that of AIDP group at 3 month and 6 month follow-up(P<0.001). The aforementioned results speculated that T-B cell interactions which are known to induce excessive humoral immune response in some subtypes of GBS may be the fundamental aspect of immunopathogenesis in the severe cases.Follicular helper T(Tfh) cells were first identified as a large population of CXCR5+CD4+T cells that predominantly localized within lymphoid follicles.CD45RO+/CD45RA- has been thought to be their memory markers. These cells accounted for(11.9±0.38) % of all CD4+T cell ’s numbers in healthy human blood,wherein the CD4+CXCR5+ fraction could be subdivided into Tfh1(CXCR3+CCR6-),Tfh2(CXCR3-CCR6-) and Tfh17(CXCR3-CCR6+)subsets. Tfh cell help to B cells is a fundamental aspect of humanal immunity, and their three subsets act as various messengers in B-cell maturation, terminal differentiation of B cells into antibody-producing plasma cells, and isotype switching. As is already known, all the changes in Tfh cells including their amount, the molecular expression and the distribution of their subsets had a robust association with the pathogenesis of several autoimmune diseases that manifested abnormal humoral immune system. At present,the research of Tfh cells in the pathogenesis of GBS has not been reported.Objective: Our study divided GBS into two major subtypes with high incidence:AIDP and AMAN. In order to study the frequency, the phenotypic characterization, the distribution of circulating memory Tfh cell subsets(Tfh1, Tfh2 and Tfh17) in the precession of the two subtypes of GBS. Then explore the potential role of the changes of the cell subsets in the early stage of the disease and find the effective regulatory pathway of humoral autoimmune as well as further investigate Tfh as proper therapeutic targets and strive to provide new ideas for personalized treatment.Methods: Thirty six newly diagnosed cases of GBS were enrolled in the study,including the 7 patients with more serious clinical signs(HFGSs≥4) for post-treatment data. Eighteen age- and sex-matched healthy individuals served as controls in this study.Disease severity was evaluated using the Hughes Functional Grading Scale(HFGS)score, and the Medical Research Council(MRC) sum score. Peripheral blood mononuclear cells(PBMCs) isolation and flow cytometry were used to explain the surface markers expression of circulating memory Tfh cells(CXCR5/CCR6/CXCR3/ICOS/PD-1) and B cells(CD19/CD27/CD38/CD20). To validate the function of Tfh subset on the differentiation of B cells and the autoantibodies production, we used flow cytometry sorting technology and then built a Tfh-B co-culture system. Enzyme-linked immunosorbent assay(ELISA) were carried out for IL-21 levels in peripheral blood, cerebrospinal fluid and co-culture supernatant.Cytometric Bead Arrays(CBA) were carried out for the levels of IL-17 A,IFN-γ,TNF,IL-10,IL-6,IL-4,IL-2 and the concentration of Ig G/Ig A/Ig M. Immunoglobulin and total protein content in cerebrospinal fluid were detected by immune scatter turbidimetry.Results: 1. A total of 36 cases of GBS were enrolled in the study, out of which 24 and 12 patients were diagnosed with AMAN and AIDP, respectively. We found that almost all of the patients with severe HFGS score(≥4) belonged to the AMAN group. 2.The frequency of circulating memory Tfh cells(CD4+CXCR5+CD45RA-) in AMAN group was significantly higher than that in the CS and AIDP groups. As to the three subsets, the absolute counts of Tfh1(CXCR3+CCR6-), Tfh2(CXCR3-CCR6-) and Tfh17(CXCR3-CCR6+) cells were all raised to a higher level in the AMAN group. However,not only the total number but also the subsets counts of circulating memory Tfh cells were no significant difference in AIDP compared with CS. 3. The percentages of memory Tfh2 and Tfh17 cells were higher in AMAN group, when compared with AIDP and CS, and the ratio of(Tfh2+Tfh17)/Tfh1 had a positive correlation with the severity of disease and the absolute plasmablasts count in blood. 4. The percentage of ICOS+cells in memory Tfh2 and Tfh17 subsets was significantly higher than that in the CS and AIDP group. Inversely, the percentage of PD-1+ cells in memory Tfh2 and Tfh17 cells in the AMAN group was found to be statistically lower when compared with that in the CS. 5. In vitro co-culture system, compared with CS, the percentages of viable B cells(CD3-CD4-) and the percentage of plasmablasts(CD38+CD20-) within B cells at day 6,were all raised to a higher level in the case group, with a concomitant increase in concentration of Ig M and Ig G in co-culture supernatants. 6. After treatment with IVIg,single dose of 0.4g/kg body weight per day for 5 consecutive days and during the plateau phases(15–32 days after onset), the frequency of total memory Tfh cells dropped, with a concomitant decline in the ratio of(Tfh2+Tfh17)/Tfh1 and serum IL-21 levels. Among them Tfh17 decreased the most obviously, followed by Tfh1, while Tfh2 did not significantly decrease. 7. The percentages of ICOS+ and PD-1+ cells in memory Tfh subsets after treatment, significantly decreased indicators only include ICOS+ cells in Tfh1 and ICOS+ and PD-1+ cells in Tfh2. 8. The number of plasmablasts(CD3-CD4-CD38+CD20-) in blood were similar in the same severe patient, before and after IVIg treatment.Conclusion: 1. This is the first study to demonstrate the association of circulating memory Tfh cells, especially their frequency, the expression of the surface markers and the skewing distribution of the three subsets with the development of GBS, which indicates that patients with AMAN not only have increased numbers of circulating memory Tfh2 and Tfh17 subsets, but are also over-activated, which is consistent with the abnormal humoral immune response. 2.The functional in vitro experiments in our study verified that memory Tfh2 and Tfh17 subsets in AMAN patients possessed stronger activity with regards to promoting B cells differentiation and secreting various autoantibodies. 3. A similar percentage of plasmablasts between pretreatment and posttreatment indicates a less than satisfactory efficacy of traditional IVIg in affording symptom-relief in patients with severe AMAN, probably due to the poor modulation of the over-activation of memory Tfh2 and Tfh17 cells. Looking for effective ways to inhibit the memory Tfh2 and Tfh17 subsets cells, and then to effectively control the over-activation of Tfh-B cells axis in the pathological state, may have future application as new therapeutic means or an adjunctive therapy with IVIg for the treatment of severe GBS patients.