Research ArticleNK CELLS

Human natural killer cells mediate adaptive immunity to viral antigens

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Science Immunology  10 May 2019:
Vol. 4, Issue 35, eaat8116
DOI: 10.1126/sciimmunol.aat8116
  • Fig. 1 Spleens and livers of BLT mice harbor tissue-specific human NK cells.

    Human NK cells were isolated from BLT mice and evaluated by flow cytometry in single-cell suspensions. Murine cells were excluded using an antibody to mouse CD45. (A and B) The frequency of human NK cells expressing the indicated transcription factor (A) or cell surface marker (B) is shown for spleens and livers of naïve BLT mice. Gating was performed on live cells by forward- and side-scatter areas, single cells, human CD45+/murine CD45 cells, and then CD56+/CD3 cells. Five genetically unrelated human donor cohorts (A) and six to seven genetically unrelated human donor cohorts of four to nine BLT mice (B) were analyzed 4 to 11 months after transplantation, with the exception of CX3CR1, for which four genetically unrelated human donor cohorts were analyzed. Unpaired t test with Welch’s correction: (A) *P = 0.05, **P = 0.01; (B) *P < 0.03, **P = 0.015, ***P < 0.0002, and ****P < 0.0001. ns, not significant.

  • Fig. 2 Spleens and livers of BLT mice harbor human NK cells similar in phenotype to human adult tissue-matched NK cells.

    Human NK cells were isolated from BLT mice and evaluated by flow cytometry in single-cell suspensions. Murine cells were excluded using an antibody to mouse CD45. The frequency of human NK cells expressing the indicated cell surface marker is shown for livers (A) and spleens (B) of naïve BLT mice and is compared with liver- and spleen-derived NK cells from adult human donors. Gating was performed on live cells by forward- and side-scatter areas, single cells, human CD45+/murine CD45 cells, and then CD56+/CD3 cells. Seven to 10 genetically unrelated human donor cohorts of four to nine BLT mice were analyzed, four to 11 months after transplantation and compared with 4 to 14 genetically unrelated human donors. Adult human donor liver NK cells were obtained from transplant-quality livers, whereas adult human donor spleen NK cells were obtained from uninvolved spleens from freshly resected treatment-naïve, HIV- and hepatitis-negative newly diagnosed patients with pancreatic cancer. Each symbol represents one mouse or human donor. Unpaired t test with Welch’s correction: **P = 0.009, ***P < 0.0005, and ****P < 0.0001. ND, not determined.

  • Fig. 3 Human hepatic NK cells mediate antigen-specific and vaccination-dependent killing.

    Human donor-matched BLT mice were left naïve or were immunized by intraperitoneal and intravenous injections with recombinant HIV-Q23-17 Env (gp140/gp120)–loaded syngeneic DCs (HIV-Env). Fourteen days after the immunization, human NK cells were isolated from either naïve or HIV-Env–vaccinated human donor-matched BLT mice by flow cytometry–based cell sorting. The NK cells were cocultured with CFSE-labeled, antigen-free, Ova-loaded, UV-inactivated H1N1 PR8 influenza A– or HIV-Env–loaded syngeneic target cells at a 1:1 ratio for 6 hours at 37°C, 5% CO2, before target cell killing was determined using flow cytometry. A total of three (spleen) to four (liver) genetically unrelated human donor cohorts of five to eight BLT mice were analyzed 5 months after transplantation, and the data were pooled for (A) and (B). Two-way ANOVA with Tukey’s multiple comparisons test; ****P < 0.0001.

  • Fig. 4 Human NK cell–mediated memory is long-lived.

    Human donor-matched PBMC and blister fluids were isolated on day 3 after VZV-DTH induction and stained for indicated markers. (A and B) Frequency of human NK cells (huCD45+ CD56+, and CD3) in blister fluid of naïve (saline-injected; n = 3) skin compared with VZV-STA–injected skin (n = 11). (C) Frequency of actively degranulating, cytotoxic human NK, as judged by cell surface expression of CD107a (n = 6). (B and C) Each symbol represents one human donor. Paired t test: *P = 0.027 and **P = 0.0037.

  • Fig. 5 Human NK memory is mediated by NK cells with a hepatic phenotype.

    Human donor-matched PBMC and blister fluids were isolated on day 3 after VZV glycoprotein challenge and stained for indicated markers. (A and B) Analysis of NK-expressed markers on degranulating (CD107a+) versus nondegranulating (CD107a) NK cells in blister fluid of VZV-STA–injected skin. An example of a histogram overlay for each marker is shown in (A) for a single donor, whereas five to seven genetically unrelated human donors were individually analyzed for each marker in (B). (C) Frequency of human NK cells expressing the indicated markers indicative of a human hepatic NK cell phenotype. Six to 11 genetically unrelated human donors were individually analyzed for each marker. (D) Frequency of human NK cells expressing the indicated transcription factor master regulators indicative of a human hepatic NK cell phenotype. Six genetically unrelated human donors were individually analyzed for each marker. Paired t test: *P = 0.05, **P = 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaat8116/DC1

    Fig. S1. Reconstitution efficiency of BLT mice.

    Fig. S2. Phenotypically mature human NK cells develop in BLT mice.

    Fig. S3. Human NK cells respond to vaccination of BLT mice.

    Fig. S4. Trans-presented human IL-15 expands NK cell numbers in spleens and livers of BLT mice.

    Fig. S5. Pre- and post-sort analyses of liver NK cells from HIV-Env–vaccinated BLT mice sorted for killing assays shown in Fig. 4.

    Table S1. Reagents used for cell surface and intracellular staining for CyTOF analysis.

    Table S2. Raw data (Excel).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Reconstitution efficiency of BLT mice.
    • Fig. S2. Phenotypically mature human NK cells develop in BLT mice.
    • Fig. S3. Human NK cells respond to vaccination of BLT mice.
    • Fig. S4. Trans-presented human IL-15 expands NK cell numbers in spleens and livers of BLT mice.
    • Fig. S5. Pre- and post-sort analyses of liver NK cells from HIV-Env–vaccinated BLT mice sorted for killing assays shown in Fig. 4.
    • Table S1. Reagents used for cell surface and intracellular staining for CyTOF analysis.

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