Clinical And Experimental Research Of The Alloreactive Natural Killer Cell In Allogeneic Hematopoietic Stem Cell Transplantation

Objective1. To explore the impact of donor killer immunoglobulin-like receptors(KIR) and donor/recipient HLA ligands on outcome in haploidentical hematopoietic stem cell transplantation(HSCT).2. To explore the impact of donor KIR and recipient HLA ligands on outcome in HLA-matched, sibling donor HSCT.3. To investigate the correlation of four types of inhibitory receptors (CD158a, CD158b, NKB1 and CD94/NKG2A) on NK cells and T lymphocytes from grafts and recipients peripheral blood cells 1m after allogeneic HSCT (allo-HSCT) with hematopoietic reconstitution and acute graft-versus-host disease(aGVHD).4. To study antileukemia effect by alloreactive NK cells mediated in murine undergoing haploidentical bone marrow transplantation(BMT).5. To investigate the promotion of immune reconstitution of alloreactive NK cells in murine undergoing haploidentical BMT.Methods1. Donor KIR and donor/recipient HLA-Cw genotype was determined by PCR -sequence-specific primer analysis(PCR-SSP).2. Four types of inhibitory receptors on NK cells and T lymphocytes were analyzed the expression by flow cytometry.3. Murine NK cells and alloreactive NK cells were prepared by immunomagnetic separation.4. The murine haploidentical BMT model was established by using C57BL/6×BALB/c (BCF₁) mice as donor, and BALB/c mice bearing EL9611 leukemia as recipient, and GVL effect was observed by infusion of grafts with or without alloreactive or non-alloreactive NK cells.5. The effect of alloreactive NK cells on immune reconstitution was observed in C57BL/6×BALB/c (BCF₁)→BALB/c murine haploidentical BMT model.Results1. In haploidentical HSCT, the presence of donor KIR2DS3, KIR3DS1 delayed neutrophil reconstitution, and KIR3DS1 delayed platelet reconstitution. KIR2DS1, KIR2DS3 and KIR3DS1 reduced cGVHD. The presence of recipient HLA-Bw4 increased aGVHD (P<0.05, respectively). Hematopoietic reconstitution, disease-free survival(DFS) and relapse were not significantly different between those with and without HLA ligands incompatibility, and those with and without KIR receptor-HLA ligand incompatibility(P>0.05, respectively).2. In HLA-matched sibling HSCT, the presence of donor KIR2DS5 delayed neutrophil reconstitution. KIR2DS2 significantly increased aGVHD and CMV(P<0.05). The incidences of cGVHD and septicemia /pneumonia were significantly lower in patients homozygous for group C1 allele than in those heterozygous for group C1/C2 allele(P<0.05). The DFS was significantly higher in patients with KIR receptor-HLA ligand incompatibility than in patients without(P<0.05), and the former group had a lower relapse trend (P=0.08).3. The percentage of CD158a, NKB1 and CD94/NKG2A expressing on NK cells in grafts was significantly higher in grade II～IV aGVHD group compared with in grade 0^I aGVHD group. The percentage of CD94/NKG2A expressing on CD8-positive T cells in grafts was significantly higher in grade 0～I aGVHD group compared with in grade II～IV aGVHD group( P<0.05, respectively).4. Alloreactive NK cells significantly prolonged survival of BALB/c mice bearing leukemia that accepted haploidentical BMT and reduced relapse (compared with nonalloreactive NK cells and no NK cells, P<0.05, respectively).5. Alloreactive NK cells significantly promoted peripheral T cell reconstitution and induced Th1 immune response in vivo in BALB/c mice received haploidentical BMT. Conclusion1. In haploidentical HSCT, the presence of donor activating KIR2DS3, KIR3DS1 delays hematopoietic reconstitution. KIR2DS1, KIR2DS3 and KIR3DS1 reduces cGVHD. The presence of recipient Bw4 increases aGVHD.2. In HLA-matched sibling HSCT, the presence of donor activating KIR2DS5 delays hematopoietic reconstitution. KIR2DS2 increases incidence of aGVHD and CMV viremia. The incidence and severity of cGVHD are significantly reduced in patients homozygous for group C1 allele. KIR receptor-HLA ligand incompatibility may reduce relapse and improve DFS.3. The higher percentage of CD158a, NKB1 and CD94/NKG2A expressing on NK cells in grafts increases grade II～IV aGVHD. The higher percentage of CD94/NKG2A expressing on CD8-positive T cells reduces grade II～IV aGVHD.4. Alloreactive NK cells significantly prolong survival of BALB/c mice bearing leukemia that accepted haploidentical BMT , and reduces relapse and has a graft versus leukemia(GVL) role.5. Alloreactive NK cells significantly promote peripheral T cell reconstitution and induce Th1 immune response in vivo in BALB/c mice received haploidentical BMT.