Members of the killer immunoglobulin family (KIR) and their HLA class I ligands contribute to establishing natural killer cell reactive potential. NK cells bearing inhibitory KIR that bind self-HLA are termed “licensed” and are highly responsive to targets lacking self HLA, but tolerant to autologous, healthy cells. Among KIR:HLA partnerships, KIR3DL1 and HLA-Bw4 alleles demonstrate the greatest polymorphism. Allelic subgroups of KIR3DL1 are distinguished by their density on the surface of NK cells and demonstrate different sensitivity to inhibition by HLA-Bw4 allotypes. Specifically, KIR3DL1 alleles expressed with high surface density (3DL1high) are more potently inhibited by HLA-Bw4 epitopes possessing isoleucine (80I) compared with threonine (80T) at position 80. It is not currently known whether the same KIR-HLA interactions that mediate higher inhibitory response also endow higher effector capacity. Retrospective analyses of patients with HIV have demonstrated that the same allelic combinations of HLA-B and KIR3DL1 predictive of high inhibition are associated with delayed progression to AIDS. Indeed, a potential target for NK cells is created by the HIV nef protein, which mediates downregulation of HLA-B expression. Taken together, these finding have led us to hypothesize that the sensitivity of NK cells for inhibition by HLA predicts the extent to which they may be licensed for functional responsiveness.

We undertook functional analyses of NK cells exclusively expressing KIR3DL1 from healthy HLA-Bw4+ or Bw4-/- donors, using HLA-negative 721.221 target cells to examine licensing function. First, we developed and validated a multiplex PCR array allowing identification of allelic groups of KIR3DL1 that correspond to expression densities. This genomic analysis informed the division of 59 subjects into groups stratified by the inhibitory potential conveyed by self HLA-B and KIR3DL1+ alleles. As expected and consistent with NK education or “licensing” by self-specific inhibitory KIR with cognate HLA ligand, KIR3DL1+Bw4+ NK cells demonstrated greater responsiveness 721.221 cells compared with KIR3DL1+ cells obtained from Bw4-/- donors, as assessed by CD107α externalization. The proportion of KIR3DL1+ NK cells degranulating in response to HLA class I-negative target cells was highly variable and not reflective of either HLA-Bw4 nor KIR3DL1 allele groups alone; however, when both KIR3DL1 and Bw4 allele groups were considered, highly inhibitory allotype pairs were indeed associated with higher NK effector function (p=0.0065). This finding was particularly pronounced among partnerships involving high-density KIR3DL1 alleles: compared with 80T, 80I conditioned 3DL1high NK cells for superior 721-221-stimulated degranulation (p=0.0035). We further investigated whether NK cell licensing could be mediated and/or maintained by HLA intrinsic to NK cells. We found that diminution of HLA expression in licensed KIR3DL1+ by shRNA-mediated knockdown reduced responsiveness to HLA class I-negative target cells, demonstrating that HLA is required on NK cells to maintain their licensed potential.

Collectively, these findings reveal that an NK cell’s capacity for effector response is not only determined by the presence of a self-specific receptor, but that functional hierarchies exist among NK cells bearing different allotypes of one KIR for its ligand in a manner correlated with inhibitory capacity. Finally, cis-interactions between KIR and HLA contribute to NK licensing, indicating that human NK education is at least partly determined by molecules intrinsic to the cell itself. These findings of differential NK licensing among KIR3DL1-Bw4 allotype combinations now provide the biological basis for the clinical findings of variable HIV control among patients with different KIR3DL1-Bw4 allotype combinations.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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