Natural killer (NK) cells can recognize virus-infected and stressed cells1 using activating and inhibitory receptors, many of which interact with HLA class I. Although early studies also suggested a functional impact of HLA class II on NK cell activity2,3, the NK cell receptors that specifically recognize HLA class II molecules have never been identified. We investigated whether two major families of NK cell receptors, killer-cell immunoglobulin-like receptors (KIRs) and natural cytotoxicity receptors (NCRs), contained receptors that bound to HLA class II, and identified a direct interaction between the NK cell receptor NKp44 and a subset of HLA-DP molecules, including HLA-DP401, one of the most frequent class II allotypes in white populations4. Using NKp44ζ+ reporter cells and primary human NKp44+ NK cells, we demonstrated that interactions between NKp44 and HLA-DP401 trigger functional NK cell responses. This interaction between a subset of HLA-DP molecules and NKp44 implicates HLA class II as a component of the innate immune response, much like HLA class I. It also provides a potential mechanism for the described associations between HLA-DP subtypes and several disease outcomes, including hepatitis B virus infection5,6,7, graft-versus-host disease8 and inflammatory bowel disease9,10.
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This work has been funded in part by the Pathogenesis and the Viral Latency Programs of the Heinrich Pette Institute, Leibniz Institute for Experimental Virology and the German Center for Infection Research (DZIF) through TTU 04.810. This project has been funded in part with federal funds from the Frederick National Laboratory for Cancer Research, under contract no. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. This research was supported in part by the Intramural Research Program of the National Institutes of Health, Frederick National Laboratory, Center for Cancer Research. W.F.G.-B. was supported by National Institute of General Medical Sciences (T32GM007752) and the NIH (P01-AI104715 and F31AI116366). P.J.N. was supported by NIH U19 NS095774. A.H. was supported by the German Center for Infection Research (DZIF) through an MD/PhD Stipend (TI 07.002) and via the Clinician Scientist Program of the Faculty of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. J.R was supported by an Australian Research Council Laureate Fellowship (FL160100049) and R.B was supported by a Career Development Fellowship from the National Health and Medical Research Council of Australia (APP1109901). We would like to thank H. Reid and K. Loh for their kind gift of HLA-DQ2 viral stocks. We would like to thank the NIH Tetramer Core Facility for all provided HLA class II monomers.
A.N., W.F.G.-B. and M.A. filed a patent application (EP18174760.1) regarding the therapeutic use of anti-NKp44 antibodies for the treatment and/or prevention of graft-versus-host disease. M.O. and J.-H.L. are current employees of OneLambda Inc., a part of Thermo Fisher Scientific. All other authors declare no competing interest.
Peer review information: Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Integrated supplementary information
Supplementary Figure 1 KIR3DL1 and KIR2DL4 Fc constructs exhibit unspecific binding to HLA class II coated beads at high concentrations.
a Binding of KIR3DL1 Fc construct at various concentrations (1–100 µg/mL) to HLA-DR (blue), HLA-DQ (yellow) and HLA-DP (red) coated beads as well as positive (grey) and negative (black) control beads is plotted as median fluorescence intensity (MFI). Each dot represents the measured MFI for binding of the KIR3DL1 Fc construct at a specific concentration to a certain HLA class II coated bead or negative/positive control beads. Lines connect matching MFI values for one specific HLA class II allotype or negative/positive control beads measured at different KIR3DL1 Fc construct concentrations. Data is representative for one single experiment (n = 1). b MFI values of KIR2DL4 Fc construct binding at varying concentrations (1–100 µg/mL; left panel) and at 10 µg/mL to HLA-DR (blue), HLA-DQ (yellow) and HLA-DP (red) coated beads as well as positive (grey) and negative (black) control beads (right panel) are depicted. Each dot in both panels represents one HLA class II molecule. Lines in the left panel connect matching MFI values for one specific HLA class II allotype or negative/positive control beads measured at different KIR2DL4 Fc construct concentrations. Horizontal line in right panel indicates median, error bars indicate interquartile range. Data is representative for one single experiment (n = 1).
Supplementary Figure 2 Untransduced Jurkat cells do not show functional responses to HLA class II molecules.
Activation of untransduced Jurkat cells in response to anti-KIR2DL3, anti-NKp46, anti-NKp44 as well as HLA-DR7 and HLA-DP401 CLIP monomers was assessed by the expression of CD69. Plot represents one of eight independent experiments (left panel). The percentage of CD69+ cells following incubation on non-coated wells (blank) was subtracted from all samples. Corrected values are illustrated as median with interquartile range as determined in eight independent biological replicates (n = 8).
Supplementary Figure 3 Unstimulated NK cells do not express NKp44 and do not degranulate upon co-incubation with HLA-DP401.
a Surface expression of NKp44 was determined in freshly isolated untreated (black) and IL-2 plus IL-15 treated (red) primary NK cells. Plot represents one of seven individual donors. MFI of NKp44 expression from untreated and cytokine-treated primary NK cells was determined in seven individual donors (n = 7). Each dot represents one donor. Horizontal line indicates the median, error bars display interquartile range. Two-tailed Wilcoxon matched-pairs signed rank test was used to assess differences in the surface expression of NKp44 between untreated and cytokine-treated primary NK cells. *p = 0.02. b Freshly isolated unstimulated NK cells were isolated from seven individual donors and co-incubated with plate-coated anti-NKp44, HLA-DR7 CLIP, HLA-DP401 CLIP or non-coated wells (blank) in the presence of purified mouse IgG1 isotype or purified anti-human NKp44 antibody (both at a final concentration of 10 µg/mL). The percentage of CD107a+ cells was determined. Each dot represents one individual donors (n = 7) and lines connect responses from one individual donor.
Supplementary Figure 4 HLA-DP surface expression of JE6.1-DP transduced cell lines is increased by CLIP pulsing.
a HLA-DP surface expression of HLA-DP transduced cell lines (red and blue histograms) is depicted. The HLA-DP expression of the untransduced parental cell line is displayed in grey. Plots represent one of seven individual experiments. b HLA-DP (left panel) and CLIP (right panel) surface expression following CLIP pulsing in comparison to DMSO-treated cells is depicted as fold change in MFI [MFI CLIP pulsed/MFI DMSO] for the four indicated JE6.1-DP expressing cell lines. Each dot represents one individual biological replicate as determined in seven independent experiments (n = 7). Boxes represent 25th to 75th percentiles. Whiskers indicate minimum and maximum values, horizontal line indicates the median.
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Niehrs, A., Garcia-Beltran, W.F., Norman, P.J. et al. A subset of HLA-DP molecules serve as ligands for the natural cytotoxicity receptor NKp44. Nat Immunol 20, 1129–1137 (2019) doi:10.1038/s41590-019-0448-4
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