Donor’s NK Cells May Influence the Engraftment in Pediatrics Patients after T-Cell Depleted Haploidentical Stem Cell Transplant for Thalassemia

In haploidentical hematopoietic transplantation, donor-versus-recipient NK cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory Killer Cell Ig-like Receptors (KIRs) for self HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated. The infusion of alloreactive NK cells

1. ablates recipient T cells which reject the graft, and

2. ablates recipient dendritic cells (DCs) which trigger GvHD, thus protecting from GvHD (Ruggeri et al., Science 2002).

NK cell alloreactivity also boosts very rapid rebuilding of donor adaptive immunity to infections. In this study we analysed the potential role of NK cells after haploidentical transplant in b-thalassemia patients.

T and B cell depletion was carried out with CD34+ coated magnetic microbeads and the CliniMACS device (Miltenyi Biotec©) from peripheral blood and bone marrow of donors (the mothers) and resulted in grafts consisting of stem cells and effector cells (NK cells, monocytes) with the addition of bone marrow mononuclear cells (BMMNCs 3 × 10⁵/kg of the recipient). A total of 11 pediatric patients with b-thalassemia received T and B cell depleted transplants from their haploidentical mothers with a median number of 15 ×106 CD34 stem cells. To analyse the mechanisms involved in immunological reconstitution post transplant, we analysed T cell subsets by flow cytometry, particularly NK sets (CD3- CD56+, CD3− CD16+ and CD56+CD16+ NK cells) at day + 20 and + 60 post transplant.

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), 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 CD3-CD56+^bright cells. Interestingly, a direct correlation has been observed between the percentages of CD56+CD16+ NK subset and the BM engraftment (in mean 71 ± 21% CD56+CD16+ in the four patients with full engraftment, 27 ± 28% in the three patients with a stable mixed chimerism after BM transplant (70–80% of donor cells) and 1.4 ± 1% in the four patients with rejection). In all the patients the origin of the NK subsets was from the mothers. 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%), in parallel with the increase of the CD3-CD16+ NK cells (potent cytotoxic effector cells) especially in the patients with full engraftment (in mean 47 ± 20% vs. 28 ± 31% in mixed chimerism)

NK CD56+^bright cells develop more rapidly than other lymphocytes, but CD16+ NK cells (with cytotoxic potential) require more prolonged exposure to maturation factor (IL-2) in the bone marrow. Interestingly we observed higher percentages of NK subsets just twenty days post transplant in the patients with full engraftment respect the mixed chimerism and the rejection, suggesting a role of donor NK cells on improved engraftment and on prevention of the rejection with the attack of the host lympho-hematopoietic cells. These observations may suggest the importance of NK subsets analyses at the first time of the transplant as an useful parameter for the outcome of the transplant and/or the use of donor’s alloreactive NK cells especially in haploidentical recipients.