Immunophenotypic, functional and gene evaluation of B cell subsets in patients with systemic sclerosis undergoing autologous hematopoietic stem cell transplantation

Author(s)

João Rodrigues Lima Júnior

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-11-11

Examining board members

Kelen Cristina Ribeiro Malmegrim de Farias; Rodrigo Alexandre Panepucci

• Advisor: Kelen Cristina Ribeiro Malmegrim de Farias; Maria Carolina de Oliveira Rodrigues

Abstract

Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease, which is characterized by microvascular changes and different degrees of skin and multiple organ fibrosis. Autologous hematopoietic stem cell transplantation (AHSCT) is currently the only treatment capable of promoting long-term clinical remission in SSc patients without the need for additional therapies. However, the immunological mechanisms involved in this therapy are not yet fully understood, including the role of B cell subpopulations. In this context, in this work we evaluated the quantitative and functional reconstitution of conventional and regulatory B cell subpopulations (Bregs) in patients with SSc treated with AHSCT (n=24). The following peripheral blood B cell subpopulations were analyzed by flow cytometry, before and after 30, 60, 120, 180 and 360 days after AHSCT: CD19+IgD+CD27- (naive), CD19+CD27+IgD+ (memory with class switch), CD19+CD27+IgD- (memory without class switch), CD19+IgD-CD27hi (plasmócito), CD19+IgD-CD27- (memory double negative), CD19+CD38-IgD+ (Bm1, naive), CD19+CD38+IgD+ (Bm2, activated naive), CD19+CD38hiIgD+ (Bm2\', pre-germinative center), CD19+CD38hiIgD- (Bm3+4, centroblasts+centrocytes), CD19+CD38+IgD- (early memory Bm5) e CD19+CD38-IgD- (late memory Bm5,), CD19+PD1+, CD20+CD27+CD43+CD69- (B1 cells), CD19+CD24hiCD38hi (transitional Breg), CD19+CD24hiCD27+ (B10 Breg). After transplantation, patients with SSc had a significant decrease in modified Rodnan skin score, serum reactive C protein and anti-Scl70 autoantibody. Six patients had disease reactivation during the post-transplant follow-up. Naive B cells were eliminated by the conditioning regimen and 360 days after transplantation showed hier absolute number than those found in the pre-transplantation period. The absolute number of memory B cells decreased transiently, returning to baseline 360 days after transplantation. In parallel, early after transplantation, we observed increased expression of PD-1 in B cells, transient increase in serum BAFF levels, and transient decrease in serum APRIL levels. Reactivated patients had increased PD-1+ B cell numbers early after transplantation. We observed a significant increase of transitional Bregs in patients who responded to AHSCT compared with those who reactivated the disease. Bregs B10 increased transiently the IL-10 production after AHSCT, while transitional Bregs showed a persistent increase of IL-10 production throughout the follow-up. Corroborating these findings, the phosphorylation of intracellular proteins ERK1/2 and p38 MAPK, which is related to B-cell IL-10 production, increased after transplantation, while STAT3 phosphorylation remained unchanged. In addition, transitional Bregs have recovered the ability to suppress CD4+ T cell proliferation, as well as the production of TNF-α and IFN-γ by these cells. On the other hand, TGF-β production by mature B cells decreased transiently, while IL-6 production persistently decreased throughout the post-transplant follow-up. We also observed a modulation of expression of 13 genes related to inflammatory response and autoimmunity 360 days post-AHSCT, compared to the pre-transplant period. Altogether, we suggest that these mechanisms act synergistically in restoring immune self-tolerance and decreasing iv inflammation and fibrosis in SSc patients who respond satisfactorily to AHSCT. The results obtained in this work contributed to increase the knowledge about the numerical and functional reconstitution of the B cell compartment after AHSCT in patients with SSc. This knowledge may contribute in the future to improvements in the clinical protocol of AHSCT for SSc and other autoimmune diseases. (AU)

• FAPESP's process: 16/24443-7 - Immunophenotypic, functional and gene evaluation of B cell subsets in patients with systemic sclerosis undergoing autologous hematopoietic stem cell transplantation
• Grantee: João Rodrigues Lima Júnior
• Support Opportunities: Scholarships in Brazil - Doctorate