Immuno Haematological Reconstitution after T-Cell-Depleted HLA-Haploidentical Stem Cell Transplantation for Thalassemia.

Background.

We evaluated haematological and immunological characteristics of four thalassemia patients after T-cell-depleted HLA-haploidentical stem cell transplantation

Methods.

We evaluated the clonogenic capability by the colony forming cell assay (CFC) and the long term culture-initiating cell (LTC-IC) assay at baseline and 20 days after transplant. Stromal cells were obtained from long term culture of bone marrow mononuclear cells (BMMCs) and analysed by immunohystochemistry. Lymphocyte subsets were studied by flow cytometry; and stromal IL-7 production by BMMCs was analysed by ELISA.

Results.

At baseline, no significant differences were observed in haematological and in immunological parameters in thalassemia patients when compared with a group of normal subjects

Day + 20 after transplant, a reduced clonogenic capability was observed (4 ± 2 vs. 41 ± 40 CFU-E, 17 ± 9 vs. 109 ± 22 BFU-E, 3 ± 1 vs. 9 ± 6 CFU-GEMM and 16 ± 10 vs. 66 ± 23 CFU-GM). The number of primitive bone marrow (BM) progenitor cells was also decreased (1.8 ± 1.4 vs. 15.4 ± 3.6 LTC-CFC/10⁶ BMMCs). In addition, 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. Upon light microscopy examination, the majority (75%) of these cells appeared as moderately large cells, frequently rounded, with abundant cytoplasm, whereas in control subjects about 90% of the stromal cells exhibited a different morphology characterized by irregular or spindle shape and branching cytoplasmic processes (fibroblast-like).

Compared with normal subjects, thalassemia patients showed: reduction of naïve CD4+ T-cells (2 ± 0.5% vs 50 ± 10%), reduction of thymic naïve CD4+ T-cells (1 ± 0.2% vs 40 ± 12%,) and a significant increase of CD4+ cells activation markers (CD95, HLA-DR and CCR5). IL-7 receptor (CD127) expression was also significantly decreased on CD4+ T-cells and on naïve CD4+ T-cells (CD4+/CD45RA+CD62L+/CD127+). NK cells were among the first lymphocytes to repopulate the peripheral blood, and up to 70% of these cells were CD56 ^brigh whereas CD16+ NK cells were decreased.

Conclusions.

Twenty days post transplant, an impaired growth and differentiation capacity of stem/progenitor cells were observed in thalassemia patients, in parallel with an altered homeostasis of T-cells and a reduction of T-cell naïve compartment. We hypothesize that the damage of T cell compartment may be at least partially due to an altered production of new T cells starting from the haematopoietic stem/progenitor cells. CD56+ NK cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factors (IL-2) in the bone marrow. An IL7/IL7R pathway dysregulation has been also observed, possibly involving bone marrow stromal cells. In vitro studies are ongoing about the use of cytokines (IL-2, IL-7, IL-2 plus IL-7) supporting T cell development.