Killer cell immunoglobulin-like receptors and their ligands : assessment of potential diagnostic and immunotherapeutic value

Human natural killer (NK) cells constitute an important arm of the innate immune system, which participates in protection against viral infections and elimination of malignant diseases. They carry variegated receptors on their surface, with killer cell immunoglobulin-like receptors (KIR) playing a main role in the regulation of their activity. The KIR family involves inhibitory and activating members. The former interact with HLA class I molecules. Decreased or absent HLA class I expression abrogates inhibition. Binding specificities of activating members are mostly unknown (except for KIR2DS1 and KIR2DS4). However, there are abundant clinical data indirectly indicating a role for activating KIRs in antiviral and antileukemic NK cell activity.
Our studies aimed to dissect the role of activating KIRs in antiviral and antileukemic activity, and to assess their potential diagnostic and immunotherapeutic value. They were performed on the level of genotype associations, by evaluation of KIR repertoire after infectious challenge, and by assessing functionality in vitro.
In the first part, we searched for predictive markers allowing identification of HIV-infected patients able to control viral replication after structured treatment interruption (STI). Candidate factors were genes known to associate with HIV viral load in untreated patients. Therefore we studied the correlation of KIR3DL1 and its ligand HLA-Bw4, but also KIR3DS1 and SNPs (HLA-C-35 and HCP5) with the evolution of viral load after STI. Only the presence of HLA-Bw4 was significantly associated with control of viral replication. However, the predictive power of this genotype was modest.
In further studies we investigated differences of KIR expression in resting NK cells in CMV-seronegative and Ðseropositive individuals, and changes in KIR repertoire after challenging NK cells with CMV-infected fibroblasts in vitro. While resting NK cells did not differ in their KIR expression, in vitro exposure to CMV caused expansion of KIR2DL1, KIR2DL3, and KIR3DS1 uniquely in CMV seropositive donors. For KIR2DL1 and KIR2DL3, expansion occurred only in patients carrying the respective KIR ligands. The expansion of KIR3DS1 positive NK cells confirmed participation of the telomeric part of KIR haplotype B in anti-CMV activity, but was unrelated to presence of its putative ligand - HLA-Bw4.
These results encouraged us to broader studies of the function of activating KIRs. We therefore expressed a selected panel of activating KIRs separately in the NK cell line NKL, generated soluble forms of each activating KIR by connecting their extracellular domains to IgG-Fc fragments, and using these reagents to control specificity, we established a novel staining method for KIR2DS5 using commercially available antibodies. Despite functionality of expressed receptors against a mouse cell line, the presence of the KIR for which some HLA class I ligands are known did produce no detectable enhancement of cytotoxicity in cytotoxicity experiments against 721.221 cells transfected with these HLA class I ligands. Soluble forms of KIR-Fc were used to screen a panel of leukemic cell lines for the presence of potential KIR ligands by flow cytometry. The HLA class I deficient cell line K562 bound KIR2DS3-Fc and KIR2DS5-Fc, and the HLA class I expressing cell lines HEL and Namalwa bound also both these KIR-Fc on their surfaces. The binding of the latter was blockable by anti-HLA antibodies and - based on the HLA configuration of both cell lines - was independent on HLA-C. The participation of the KIR2DS5 receptor in killing of K562 cells by primary NK cells was indirectly confirmed, but requires a further investigation.
Collectively, these data suggest a potential antiviral effect of KIR3DS1 and a possible antileukemic effect of KIR2DS5. Their presence could predict individual immune responses, and taking into account their function could be beneficial in immunotherapeutic settings.