Fig 2: LIR-1+ expanded NK cells show increased responsiveness to K562 cells compared with their resting and overnight IL-2-activated counterparts. NK cell degranulation as measured by CD107a cell surface expression on resting, overnight (ON) IL-2-activated and expanded NK cells from KIR haplotype A/A donors following co-culture with K562 cells. (a) Zebra plots showing degranulation by selected NK cell subsets from one representative donor educated on the KIR3DL1 ligand HLA-Bw4. (b) Pooled data comparing degranulation by NK cell subsets either lacking all of the investigated inhibitory receptors (iNKR-) or single positive (SP) for LIR-1, educated KIR or educated NKG2A (iNKR-, n = 10; LIR-1SP, n = 10; KIR2DL1SP, n = 7; KIR2DL2/3SP, n = 7; KIR3DL1SP, n = 4; and NKG2ASP, n = 10). Paired analysis was performed using the Wilcoxon matched-pairs signed-rank test. (c) Pooled data comparing fold change in degranulation by NK cell SP-uneducated KIR (n = 10), NK cell SP-educated KIR (n = 18) and LIR-1 (n = 10) subsets over its individual matched iNKR- subset. Paired analysis was performed using the Wilcoxon signed-rank test between LIR-1SP and the matched groups of uneducated and educated KIRSP, respectively, while unmatched analysis was performed between uneducated and educated KIRSP groups using the Mann–Whitney U-test. Individual groups were compared with iNKR- using the Wilcoxon signed-rank test and comparing with a hypothetical value of 1. The education status of each KIR was determined on receptor expression and genomic presence of cognate KIR ligand. Bars show mean, and error bars represent SEM.

Fig 3: LIR-1 co-education with KIR and NKG2A on expanded NK cells is insufficient to overcome KIR- and NKG2A-mediated inhibition. (a) Degranulation as measured by CD107a cell surface expression on resting, overnight (ON) IL-2-activated and expanded NK cells following co-culture with HLA class Ilow 721.221 wild-type (WT) cells. (b) Degranulation by the denoted donor matched expanded NK cell subsets following co-culture with 721.221 wild-type (WT) or 721.221 HLA class I transfectants. Data were generated using KIR haplotype A/A donors, and only KIR-educated NK cell subsets were selected (KIR2DL1 single positive (KIR2DL1SP), n = 7; KIR3DL1SP, n = 4; NKG2ASP, n = 10; KIR2DL2/3SP, n = 7; iNKR-, n = 10; and LIR-1SP, n = 10). For each graph with HLA class I transfectants, the number of donors was reduced in iNKR- and LIR-1SP subsets to resemble only matched depicted SP subsets. Paired analysis was performed using the Wilcoxon matched-pairs signed-rank test. Bars show mean, and error bars represent SEM.

Fig 4: Selective detuning of the responsiveness by LIR-1SP expanded NK cells following 24-h antibody blockade. Degranulation levels as measured by CD107a cell surface expression on expanded NK cells either pre-blocked or not with a neutralising LIR-1-specific antibody for 24 h prior to co-culture with K562. Data represent matched donors with high responsiveness by the LIR-1 single-positive (LIR-1SP) NK cell subset against K562 and are independent of KIR education status of both KIR haplotype B/x and A/A donors. (a) Data presenting the degranulation by the iNKR- and the LIR-1 and KIR SP NK subsets in the absence and presence of LIR-1 blockade (n = 10, except for KIR3DL/S1SP that has n = 6). (b) Pooled data comparing the responsiveness of NK cell subsets expressing either LIR-1 or a KIR alone or co-expressing LIR-1 with one KIR (KIR1+LIR-1+) compared with iNKR- (iNKR-, n = 10; LIR-1SP, n = 10; KIRSP, n = 24; and KIR1+LIR-1+, n = 24). Paired analysis was performed using the Wilcoxon matched-pairs signed-rank test. Bars show mean, and error bars represent SEM.

Fig 5: LIR-1 co-expression with an educating KIR receptor increases the responsiveness of expanded NK cells. NK cell degranulation as measured by CD107a cell surface expression on resting and expanded NK cells from KIR haplotype A/A donors following co-culture with K562 cells. (a) Gated on NK cell subsets co-expressing one educated KIR and LIR-1 compared with NK cell subsets expressing only one educated KIR or the LIR-1 receptor or cells lacking inhibitory receptors (iNKR-) (KIR2DL1 graph, n = 7; KIR2DL2/3 graph, n = 7; and KIR3DL1 graph, n = 4). Paired analysis was performed using the Wilcoxon matched-pairs signed-rank test. (b) Pooled data showing fold change degranulation by educated NK cells co-expressing LIR-1 with an educating KIR compared with the corresponding NK cell subset only expressing the educating KIR (n = 18). KIR1+LIR-1+ cells were compared with KIRSP using the Wilcoxon signed-rank test and comparing with a hypothetical value of 1. Bars show mean, and error bars represent SEM.