Natural Killer (NK) cells are innate immune cells that mediate antiviral and antitumor responses. NK cell activation and induction of effector functions are tightly regulated by the integration of activating and inhibitory receptors such as killer immunoglobulin-like receptors (KIR). KIR genes are characterized by a high degree of diversity due to presence or absence, gene copy number and allelic polymorphism. The aim of this study was to establish the distribution of KIR genes and genotypes, to infer the most common haplotypes in an admixed Colombian population and to compare these KIR gene frequencies with some Central and South American populations and worldwide. A total of 161 individuals from Medellin, Colombia were included in the study. Genomic DNA was used for KIR and HLA genotyping. We analyzed only KIR gene-content (presence or absence) based on PCR-SSO. The KIR genotype, most common haplotypes and combinations of KIR and HLA ligands frequencies were estimated according to the presence or absence of KIR and HLA genes. Dendrograms, principal component (PC) analysis and Heatmap analysis based on genetic distance were constructed to compare KIR gene frequencies among Central and South American, worldwide and Amerindian populations. The 16 KIR genes analyzed were distributed in 37 different genotypes and the 7 most frequent KIR inferred haplotypes. Importantly, we found three new genotypes not previously reported in any other ethnic group. Our genetic distance, PC and Heatmap analysis revealed marked differences in the distribution of KIR gene frequencies in the Medellin population compared to worldwide populations. These differences occurred mainly in the activating KIR isoforms, which are more frequent in our population, particularly KIR3DS1. Finally, we observed unique structural patterns of genotypes, which evidences the potential diversity and variability of this gene family in our population, and the need for exhaustive genetic studies to expand our understanding of the KIR gene complex in Colombian populations.

The long-term allograft survival has been attributed to the development of operational tolerance due different mechanisms as clonal deletion, anergy and immune regulation. Previous studies have demonstrated that tolerogenic dendritic cells play a key role in the induction and maintenance of the peripheral tolerance. Therapies based on tolerogenic DCs transfer, differentiated with pharmacological agents, are a promising strategy for transplantation tolerance induction while reducing dependency on immunosuppressive drugs, however the reduced number of the circulating CD14+ cells represents a limitation for the development of the therapeutic strategies. Recently, studies have suggested that the human spleen may constitute an extramedullary reservoir of monocytes in humans. Based on this considerations, we proposed to evaluate whether the methods described to differentiate dendritic cells whit tolerogenic markers from blood monocytes in vitro are also valid in CD14+ cells derived from human spleen. Dendritic cells were differentiated from blood and spleen CD14+ cells cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 7 days and in the absence or presence of vitamin D3. By flow cytometry, we evaluated the percentage of differentiation of Lin1-HLA-DR+ Cells and surface expression of ILT3, PD-L1 and PD-L2 as markers of tolerogenic dendritic cells. The percentage of differentiated DCs was not significantly different between peripheral blood and spleen CD14+ cells cultures (75.96±16.23% vs 78.45 ±14.26%); and the presence of Vitamin D3 did not interfered with the differentiation process (79.06 ±16.06% vs 77.11±14.21%). Interestingly, when these cells were differentiated in the presence of Vitamin D3, we observed higher percentage of tolerogenic markers as demonstrated by high surface levels of ILT3, PD-L1 and PD-L2 molecules. The increased expression of these molecules were significantly both in spleen and blood cells cultures except PD-L1 which was higher only in spleen differentiated dendritic cells. We could demonstrate that the methods described to differentiate DC from blood monocytes are applicable to CD14+ cells derived from human spleen. These findings open the possibility of using spleen from deceased donors as a source of dendritic cells precursors for the development of new therapeutic treatments that permit the induction and maintenance of transplantation tolerance.