Killer cell immunoglobulin-like receptors (KIR), licensing and ectosomes in the regulation of natural killer cell function : clinical implications and perspectives

Natural killer (NK) cells represent the largest proportion of innate effector lymphocytes and account for 10-15% of total peripheral lymphocytes. In contrast to B- and T- cells, NK cell activation does not depend on clonally rearranged and antigen specific surface receptors. Instead, NK cells rely on an array of activating and inhibitory germ-line encoded receptors. When encountering a target cell, activating and inhibitory signals are integrated and a response is formed immediately. Killer cell immunoglobulin-like receptors (KIR) are thereby critical for determination of NK cell activation. KIR receptors can be either activating or inhibitory in nature. Inhibitory KIR receptors not only regulate NK cell function, but are also involved in a developmental process called “licensing”. During licensing, NK cells which express an inhibitory KIR capable of recognizing HLA develop increased functional competence. Both, inhibition of mature NK cells and NK cell licensing are based on the interaction of inhibitory KIR with their cognate HLA class I ligands encompassing HLA-C and subsets of HLA-B and HLA-A. NK cell “licensing” is still poorly understood and many questions remain open. So far it remains elusive if the quantity of HLA expression is a variable in NK cell licensing. We took advantage of two recently identified polymorphisms affecting the HLA-C expression on the cellular surface rs9264942 and rs67384697. We genotyped 66 healthy blood donors for these polymorphisms and assessed the quantity of HLA-C expression by FACS using an HLA-C specific antibody. In addition, we assessed the presence of the inhibitory KIR receptors KIR2DL1and KIR2DL3 and their corresponding HLA ligands, being two mutually exclusive groups of HLA-C: HLA-C1 and HLA-C2. In subsequent functional analyses, we observed, in agreement with the concept of licensing, increased functionality of KIR2DL1+ and KIR2DL3+ NK cells in donors expressing the corresponding HLA ligand in a dose dependent manner,. The quantity of HLA-C surface expression, however, did not affect the quality of NK cell licensing.
A unique opportunity to study NK cell licensing is provided during the first months after hematopoietic stem cell transplantation when the NK cell repertoire is rebuilt. Previous studies found that leukemia patients receiving hematopoietic stem cell transplantation (HSCT) from a haploidentical donor, benefit from a survival advantage if KIR ligands are mismatched and NK cell tolerance is irreversibly broken. Recent studies reported controversial findings concerning a survival advantage and NK cell mediated graft versus leukemia effect (GVL) as a consequence of disturbed NK cell licensing early after HSCT, when NK cells are unselectively equipped with functional capacity. Differences in allogeneic HSCT protocols were discussed as possible reason for the opposing results. We investigated reconstitution of NK cell function and NK cell licensing in 56 patients receiving allogeneic (33) or autologous (23) HSCT during the first six months after transplantation. We found that NK cell licensing was maintained after both kinds of transplantation. However licensing effects were less distinct after allogenic compared to autologous HSCT. Additionally, we identified GvHD and pre-transplant ATG administration as variables associated with less prominent licensing in recipients of allogeneic grafts.
Whereas research on inhibitory KIR and NK cell licensing has already influenced treatment modalities of HSCT, much less is known about activating KIR receptors. Ligands to many activating KIR remain unidentified and little is known in which diseases activating KIR may play a role. Recent studies in our lab and elsewhere described associations between KIR genotypes and relative protection from cytomegalovirus (CMV) replication after solid organ transplantation (SOT) and HSCT. We prospectively followed a cohort of 649 patients after SOT, assessed the KIR genotype and recorded common opportunistic viral infections. Subsequent analyses of our data revealed an association of KIR B haplotypes, which encompass many activating KIR, and relative protection from varicella zoster virus (VZV) and a tendency for relative protection from Epstein-Barr virus (EBV) replication. In subsequent analyses we found that centromeric rather than telomeric activating KIR protect from VZV. In contrast, we detected no association between activating KIR genotype and BK polyomavirus (BKPyV) or Herpes simplex (HSV) replication.
Besides intrinsic factors such as licensing or expression of activating KIR, extrinsic factors determine antitumor and antiviral activity of NK cells. Early after HSCT when NK cell driven GVL is assumed most effective, prophylactic immunosuppression is given and cellular products are administered including erythrocytes (ERY) and platelets (PLT). During storage, blood products release ectosomes. PLT ectosomes can reduce monocyte and dendritic cell function. Furthermore, they favour differentiation of naïve T-cells into T-regulatory cells. However, little is known about their interaction with NK cells. We assessed phenotypical and functional changes on NK cells after co-incubation with PLT ectosomes in vitro, and found a reduction of NK cell function and activating surface receptors, mediated through TGF-β on PLT ectosomes.