Newly-Generated Regulatory B- and T-Cells Are Associated with Clinical Improvement and Reversal of Dermal Fibrosis in Systemic Sclerosis Patients after Autologous Hematopoietic Stem Cell Transplantation
Authors: Arruda Lucas C M , Lima-Júnior João R , Clave Emmanuel , Moraes Daniela A , Douay Corinne , Fournier Isabelle , Moins-Teisserenc Hélène , Covas Dimas Tadeu , Malmegrim Kelen , Farge Dominique , Ramalho Leandra , Toubert Antoine , Oliveira Maria Carolina 
Published: October 2019

Abstract
Background: Autologous Hematopoietic Stem Cell Transplantation (AHSCT) has been shown more effective to treat severe forms of systemic sclerosis (SSc) than conventional immunosuppressive therapy.However, despite advances in clinical management, therapeutic mechanisms of AHSCT still need to be more completely understood. Furthermore, identification of biomarkers of therapeutic response may help to refine current clinical protocols and improve future therapeutic goals.
Objective:To investigate immunological mechanisms associated with the therapeutic efficacy of AHSCT.
Methods: Thirty-one patients with progressive SSc, refractory to first line therapy, were selected for AHSCT. Follow-up included clinical assessment of general health, skin thickness (modified Rodnan's Skin Score, mRSS), lung function (Forced Vital Capacity, FVC), levels of anti-Scl70 autoantibodies and C-reactive protein (CRP). Dermal fibrosis was evaluated by PicroSirius (PS), hematoxylin/eosin (HE) and Masson's-Trichrome (MT). Sixteen additional SSc patients under standard treatment were enrolled as controls. PBMCs were collected before and every 6 months, until 36 months post-AHSCT. Thymic function was measured by RT-qPCR quantification of β- and signal joint (sj)-T-cell receptor excision circles (sjTREC) and intra-thymic T-cell division (n) was calculated by the formula: n= LOG(sjTREC/βTREC)/LOG2. B cell replication history was quantified by coding-joint (Cj) and sj-kappa-deleting recombination excision circles (sjKREC), and B-cell divisions in the peripheral blood (N) were calculated by the formula: N= LOG(Cj/sjKREC)/LOG2. Telomere length was evaluated by multiplex RT-qPCR. CD19+CD24hiCD38hi Bregs, CD19+CD27-IgD+ naive, CD19+CD38lowIgD+ Bm2, CD3+CD4+CD31+CD45RA+ recent thymic emigrants (RTE), CD8+CD28-CD57+ exhausted and CD4+CD25hiFoxP3+ (GITR+/CTLA-4+) Tregs were quantified by FACS. IL-10-producing Tregs were quantified after 24h stimulation with anti-CD3/CD28 Dynabeads. IL-10-producing Breg counts were assessed after 24h stimulation with CpG and CD40L. Skin biopsies were stained for IL-10 expression.
Results: Rodnan's score decreased at 6 months, remaining lower than baseline until 36 months after AHSCT. FVC values stabilized and anti-Scl-70 autoantibody titers and C-reactive protein (CRP) levels decreased after AHSCT. PS, HE and MT skin biopsy staining evidenced decreased collagen deposition. Immune reconstitution analyses evidenced that thymic function was significantly reduced at 6 months post-AHSCT, and higher than baseline at 24 months, never changing intrathymic T-cell division rates and strongly correlating with thymic-derived RTE exportation. Moreover, at 6 months post-AHSCT, increased numbers of exhausted T-cells correlated with reduced telomere T/S ratio, indicating that while thymic function is suppressed, early after AHSCT, the immune reconstitution is based on homeostatic proliferation of residual cells. Regulatory T-cells increased at 12 months post-transplantation, correlating with sjTREC values, with higher CD45RA expression and IL-10 production than at baseline. Bone marrow output of naive B-cells increased from 12 until 36 months post-AHSCT, resulting in reduced B-cell division in the periphery, as shown bypersistent increase of conventional naive as well as of non-conventional Bm2 naive B-cell counts. Finally, regulatory B-cell counts increased transiently from 6 to 12 months after AHSCT, presenting higher IL-10 production than at baseline. Skin biopsies evidenced higher IL-10 expression at 6 and 12 months post-transplantation as compared to baseline. Six transplanted patients presented disease reactivation with subsequent increase in Rodnan's score and/or worsening of lung function after AHSCT. These patients presented lower FoxP3, GITR and CTLA-4 expressions at 12 months post-transplantation. Regulatory B-cell counts after AHSCT were also lower in relapsing than in non-relapsing patients.
Conclusions: Our results suggest that increased counts of newly-generated regulatory B- and T-cell after AHSCT are associated with clinical improvement in SSc patients. Clinical remission may be associated with amelioration of the immunoregulatory network and restoration of self-tolerance promoted by the procedure.

Disclosures
No relevant conflicts of interest to declare.