Figure 1. Schematic iNKT cell reconstitution dynamics and correlation with disease control following hHSCT in pediatric leukemia patients. A longitudinal analysis of iNKT cell reconstitution in relation to disease remission was performed monthly for 18 mo post-hHSCT in 22 children affected by leukemia and myelodisplasia. This data were integrated by a cross-sectional analysis of iNKT cell frequency and maturation in a second cohort of 11 leukemia patients that were in stable remission from 1 to 8 y after transplantation. (A) Schematic illustration of the transplantation physiology in case of HLA-haploidentical conditions. Donor CD34+ HSC are T cell depleted to reduce the risk of GVHD due to donor/recipient HLA barrier. Patients are myeloablated to eradicate malignant cells and eliminate the endogenous immune system and then infused with purified CD34+ HSC. Pro-thymocytes, originated from transplanted HSC that have reached the bone marrow, colonize the thymus and generate T and iNKT cells that are exported in the periphery. iNKT cells are detectable in periphery of patients maintaining remission as early as 3 mo post-hHSCT and reach an average of . 10 iNKT/106 T cells in the 18 mo observation time, whereas iNKT cells fail to reconstitute in relapsing patients, remaining on average , 20 iNKT/106 T cells. (B) Immunoreconstitution dynamics of human iNKT cell following hHSCT. The left panel depicts the different dynamics followed by the CD4+ and CD4- iNKT cell subsets. The CD4+ emerge before the CD4- ones in the periphery and remain the dominant subset up to 4–5 y post-transplant. The CD4- cells undergo a massive expansion around 7 y post-hHSCT, resulting in the CD4- . CD4+ iNKT cell ratio typical of adult healthy individuals. The right panel shows the dynamics of the terminal NK-maturation of the two iNKT cells subsets, as defined by the acquisition of CD161 (NKPR1a-c). Both CD4+ and CD4- subsets emerge in the periphery with an immature CD161- phenotype. The CD4- subset matures markedly more rapidly than the CD4+ cells and reaches adult CD161-expression levels already by 2 y post-hHSCT, whereas the CD4+ iNKT cells have not yet completed their NK-maturation after 7 y post-transplantation. the peripheral blood of recipients at monthly intervals up to 18 mo post- hHSCT, correlating their emergence with disease relapse and analyzing their recon- stitution and maturation kinetics using mathematical models. The data on the reconstitution dynamics of iNKT cells was further reinforced by adding a cross- sectional analysis in a second group of 11 patients, who were in clinical remission for 2–6 y post-hHSCT. Two major findings were obtained (Fig. 1B). First, we identified a clearly different population dynamics of the CD4+ and CD4- iNKT cell subsets. The CD4+ emerged first and increased in the periphery at a rate twice faster than that of the CD4- cells. Both subsets appeared with an immature CD161- phenotype; however, the CD4- cells matured markedly more rapidly than the CD4+ iNKT cells, reaching adult-like frequency of CD61 and IFNc expression already by 6 mo after their first detec- tion. The CD4+ iNKT cells remained the most abundant subset up to 5–6 y post-hHSCT, when the CD4- cells under- went a brisk expansion resulting in the CD4- → CD4+ iNKT cell ratio typical of adults. These data suggested that the making of a mature iNKT cell repertoire is a slow process regulated by subset- specific mechanisms. Second, we observed a correlation between the presence of iNKT cells and leukemia remission. iNKT cells fully reconstituted in all 14 patients maintaining remission, whereas they completely failed to reconstitute in the 8 relapsing patients. Although the number of patients analyzed was relatively small, none of the best-known variables (female-to-male combination, donor NK 356 OncoImmunology Volume 1 Issue 3