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Evaluation of immunological mechanisms associated with autologous hematopoietic stem cell transplantation in systemic sclerosis patients

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Author(s):
Lucas Coelho Marlière Arruda
Total Authors: 1
Document type: Doctoral Thesis
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Maria Carolina de Oliveira Rodrigues; Cristiane Kayser; Geraldo Aleixo da Silva Passos Junior
Advisor: Maria Carolina de Oliveira Rodrigues; Kelen Cristina Ribeiro Malmegrim de Farias
Abstract

Autologous hematopoietic stem cell transplantation (AHSCT) is more effective for patients with severe systemic sclerosis (SSc) than conventional immunosuppression (IS). However, the immunological mechanisms associated with the therapeutic efficacy of AHSCT are not fully elucidated. Peripheral blood mononuclear cells and serum/plasma were collected from 31 SSc patients before and semiannually, until 36 months post-transplant, and from 16 nontransplanted SSc patients treated with IS. Thymic function was measured by RT-qPCR quantification of ?- and signal joint (sj)-T-cell receptor excision circles (sjTREC) and intrathymic T-cell division (n) was calculated by the formula: n=LOG(sjTREC/?TREC)/LOG2. Bcell replication history and bone marrow function were assessed by coding-joint (Cj) and sjkappa-deleting recombination excision circles (sjKREC). B-cell divisions in the peripheral blood (N) were calculated by the formula: N=LOG(Cj/sjKREC)/LOG2. CD3+CD4+CD31+CD45RA+ recent thymic emigrants (RTE), CD4+CD25hiFoxP3+ (GITR+/CTLA-4+) regulatory T-cells (Tregs), CD19+CD27-IgD+ naïve B-cells, CD19+CD38lowIgD+ Bm2 B-cells, CD19+CD24hiCD38hi regulatory B-cells (Bregs), CD8+CD28-CD57+ senescent cells and PD1+ exhausted cells were quantified by FACS (fluorescence-activated cell sorting). The T-cell receptor (TCR) was sequenced by New Generation Sequencing and the profile of inflammatory and pro-fibrotic serum cytokines was evaluated by CBA-Flex (cytometric bead-array) and ELISA (enzyme-linked immunosorbent assay). sjTREC and ?TREC values decreased at 6 months post-AHSCT, returning to pretransplant values at 12 months, correlating with RTE counts and associated with higher diversity of the TCR. There was no change in thymocyte division rates. At 12 months postAHSCT, Treg counts increased and correlated with sjTREC values, presenting increased expression of GITR and CTLA-4 when compared to pre-transplant levels. From 12 months until the end of follow-up, sjKREC values increased, while those of Cj remained stable, correlating with increased counts of naïve and Bm2 B-cells, resulting in reduced rate of B-cell division. There was an increase of Breg frequency from 6-months until one-year after AHSCT, correlating with sjKREC values and presenting higher IL-10 production after stimulation with CPG±CD40L than before transplantation. Telomere length decreased at 6 months post-transplant and correlated with elevated levels of FoxP3-expressing senescent cells, together with increased expression of PD1 by T-cells and reduced serum IL-6, IL-1b and C-reactive protein levels. Six patients relapsed after transplantation, presenting lower expression of FoxP3, GITR, CTLA-4 by Tregs, decreased Breg counts and reduced TCR diversity. In addition, clinical remission was associated with increased PD1 expression by T and B cells and low serum levels of TGF-?, IL-6, IL-1, IL-17A, MIP-1, G-CSF and IL-12. Therefore, newly-generated regulatory T and B cells after AHSCT, associated with T-cell repertoire renewal, high PD1 expression and low serum levels of inflammatory and profibrotic mediators associate with clinical outcomes of SSc patients after AHSCT. (AU)

FAPESP's process: 13/18678-3 - Study of immunological mechanisms involved in the therapeutic response of patients with systemic sclerosis to autologous hematopoietic stem cells transplantation
Grantee:Lucas Coelho Marlière Arruda
Support type: Scholarships in Brazil - Doctorate