Early T Cell Recovery of Thymus-Derived Naive T Cells and NK Cells in Pediatrics Patients after T-Cell Depleted HLA-Haploidentical Stem Cell Transplantation for Thalassemia.
Authors: Isgro’ Antonella , Sodani Pietro , Marziali Marco , Erer Buket , Alfieri Cecilia , Gaziev Javid , Andreani Marco , Leti Wilma , Campagna Massimo , Aiuti Fernando , Lucarelli Guido 
Published: April 2020

Abstract
Delayed immune recovery post transplant remains a significant obstacle and results in increased risk of infections. T cells are regenerated via 2 pathway, thymus-derived and peripheral expansion, processes for which IL-7 is critical. To analyse the mechanisms involved in immunological reconstitution, we studied six thalassemia patients after 20 and 60 days post T-cell-depleted HLA-haploidentical stem cell transplantation. The mean age ranged from 14 to 5 years. As controls, 6 healthy donors matched by sex and age with the patients were included. We analysed T cell subsets by flow cytometry. Stromal cells, obtained from long term culture of bone marrow mononuclear cells were analysed by immunohystochemistry and the stromal IL-7 production was analysed by ELISA. Day + 20 post transplant, the patients had significantly lower CD4+ T cells in comparison to the controls (1.9 ± 1.4% vs. 47.5 ± 6% respectively), and this reduced number was mainly observed in CD45RA+CD62L+ (naive phenotype) subset (1.3 ± 2% in patients vs. 52 ± 12% in controls). A significant decrease of peripheral CD45RA+CD31+ Th cells (thymic naive Th cells) (on average 0.5 ± 0.3% in patients vs. 37 ± 10% in controls) was observed, whereas CD8+ T cells numbers did not statistically differ between patients and controls (24.2 ± 33.7% vs. 20 ± 7%). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD56 bright whereas CD56dim CD16+ NK cells were reduced. Day + 60 post transplant an increase in the percentages of CD4+ T cells, naïve CD4+ cells and in thymic naïve Th cells were observed (3 ± 1.2%, 2.9 ± 2.1%, 2.7 ± 1%, respectively). CD8+ T cells were also increased (in mean 35 ± 27.5%). Compared with normal subjects, thalassemia patients showed a significant increase of CD4+ cell activation markers (CD95, HLA-DR and CCR5) and this was observed after 60 days post transplant, in parallel with the increase of the CD56dim CD16+ NK cells especially in the patients with full engraftment. Stromal cells secreted lower IL-7 levels (0.3 + 0.1 pg/mL vs. 0.8 + 0.1 pg/mL, in controls) and displayed by immunohistochemistry an altered phenotype (“macrophage-like” morphology). A significant decrease in total lymphocyte counts and depletion of CD4+ T cells expressing predominantly the CD45RA+CD62L+ phenotype were observed after 60 days post transplant. Also the CD4+CD45RA+CD31+ T cell subset was initially reduced but an increase has been observed at day + 60 post transplant, suggesting a thymus involvement in these patients. An IL7/IL7R pathway dysregulation has been also observed, possibly involving bone marrow stromal cells. NK cells were among the earliest lymphocytes to repopulate the peripheral blood, but. CD56dim CD16+ NK cells were increased after 60 days post transplant, especially in the patients with full engraftment, suggesting a role of donor NK cells on bone marrow engraftment. We hypothesize that the recovery of T cell compartment may be due to a deregulated production of new T cells starting from haematopoietic stem cells under the influence of stromal cytokines production.