KIR genotype profiling and FCyR3A gene polymorphisms in non T-cell depleted, G-CSF primed, HLA-haploidentical bone marrow transplantation.

Following haploidentical hematopoietic stem cell transplantation (HSCT),donor alloreactive natural killer (NK) cells favorably influence the outcome of transplanted patients by triggering graft versus leukemia (GvL) effects. The molecular mechanism underlying the GvL activity of NK cells is due to KIR/HLA class I disparity between donor/recipient pairs. KIR genotypes are important in controlling KIR repertoire. Furthermore, the activation of antibody-dependent cellular cytotoxicity (ADCC) mediated by polymorphic CD16 antigens may potentiate the GvL effect of NK cells. In unmanipulated non T cell depleted HSCT, we evaluated (a) KIR genotype profiling during immune reconstitution; (b) reconstitution of KIR loci on the basis of donor KIR genotypes and (c) polymorphisms of FCGR3A according to 158V/F and 48 L/H/R in 16 donor/recipient pairs. DNA and RNA were isolated from peripheral blood using spin bind method and cDNA was obtained by reverse transcription using RNA as template and random primers. KIR genotyping and gene expression profiling were performed using a KIR SSP typing kit which detects 17 KIR genes. Sequence based typing (SBT) assay was used to determine the FCGRIIIA-48 and -158 polymorphisms. Four pts developed severe acute Graft versus Host Disease (aGvHD),1pt relapsed and 8 pts deceased for GvHD, relapse or infections. Six of 16 pts had more than 2 KIR-ligand mismatches in GvH direction. Examining KIR profiles in recipient-donor pairs, we found that all framework genes (2DL4,3DL2, 3DL3,3DP1) were present in both donor and recipient cells, exhibiting at least one inhibitory KIR. During recipients hematopoietic recovery, we observed rapid appearance of donors KIR profiles. During KIR reconstitution monitoring, we noted different levels of 3DL1,2DS1,2DL2,2DL3 KIR expression and 2DS4ins,2DS2, 2DS3, 3DL3 KIR loss. The analysis of FCGRIIIA polymorphisms evidenced that 75% of recipients (n=12) and 69% of donors (n=11) had 158V/F respectively while 25% of recipients (n=4) and 31% of donors (n=5) had 158V/V respectively. Noteworthy was that none of recipients and donors showed F/F genotype, such as observed in other Italian studies (Calemma R., 2012). Regarding FCG3A-48 polymorphism, we observed in patients a frequency of 68% for L/L (n=11), 6% L/H (n=1), 19% L/R (n=3) and 6% H/R (n=1), while in donors 75% L/L (n=12), 6% L/H (n=1), 12.5% L/R (n=2), 6.0% R/R (n=1) and 0% H/R. In 9 of 12 V/F AA 158 pts, we observed a correlation with AA48L/L. These findings could help to interpret the retrospective analyses regarding the NK alloreactivity and should be taken in account to the best donor choice on the basis of the presence of a KIR ligand mismatch involving a KIR reconstituting early after transplantation.