Human NK cells of mice with reconstituted human immune system components require preactivation to acquire functional competence - PubMed (original) (raw)

Human NK cells of mice with reconstituted human immune system components require preactivation to acquire functional competence

Till Strowig et al. Blood. 2010.

Abstract

To investigate human natural killer (NK)-cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NOD-scid IL2Rγ(null) mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46(+)CD56(-) NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16(+) NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16(+) and CD16(-) NK cells efficiently produce interferon-γ and degranulate in response to stimulation with NK cell-susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer.

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Figures

Figure 1

Figure 1

Distribution of human NK cells in reconstituted NSG mice. Frequency within human CD45 positive cells and total number of CD3−NKp46+ human NK cells in spleen, blood, lung, liver, and bone marrow (BM) of NSG mice reconstituted with human immune system components 3 months after transfer of CD34+ hematopoietic progenitor cells. Representative flow cytometric staining from 1 mouse (A) and composite data from 7 mice (B-C) are shown. Numbers in plots represent frequencies within gates.

Figure 2

Figure 2

NK-cell subset development in NSG mice in comparison to human adult and CB. CD3−CD56+ human NK cells from spleens of reconstituted NSG mice were analyzed for KIR, CD94, NKG2D, CD16, NKG2A, and NKp46 expression (A). One representative of 12 mice is shown. CD3−NKp46+ human NK cells from spleens of reconstituted NSG mice were analyzed for CD56, CD16, and NKG2A expression (B). A representative staining from 1 of 12 mice is shown. CD16, KIR, CD127, and CD117 expression on CD3−NKp46+CD56− cells of hu-NSG mice was analyzed (C). One representative of 3 stainings is displayed. In comparison, CD56 and CD16 expression was analyzed on CD3−NKp46+ NK cells from human adult (PB) and CB. One representative example (D) and composite data (E) of 8 experiments are shown. CFSE-labeled CD3−NKp46+CD56− cells of hu-NSG mice were adoptively transferred into hu-NSG mice, which had been reconstituted with autologous CD34+ HPCs. CD56 expression on the recovered NKp46+ CFSE-labeled cells from recipient spleens was analyzed after 48 hours by flow cytometry (F). Gray shaded histogram of post-sort CD3−NKp46+CD56− cells in comparison to black histogram of post-sort CD56+ (left panel). White histogram of recovered cells in comparison to gray shaded histogram of cells that were transferred (right panel). One representative of 2 experiments is shown. Numbers in plots represent frequencies within gates or quadrants.

Figure 3

Figure 3

Functional activity of human NK cells from reconstituted NSG mice in comparison to human adult or CB NK cells. Degranulation, a surrogate marker for cytotoxicity, and cytokine production were assessed by surface CD107a and intracellular IFN-γ staining in response to ex vivo stimulation with medium, the erythroleukemia cell line K562, the T-cell lymphoma cell line CEM, and the monokines IL-12 plus IL-15 (IL-12/15). One representative staining (A) and composite data (B) of 12 mice in 3 experiments are shown. In the composite data human NK-cell reactivity of reconstituted NSG spleens was compared with 3 adult PBMC samples. In addition, the function of the human NK-cell subsets CD56brightCD16−, CD56dimCD16+ and NKp46+CD56− from human adult (PBMC), hu-NSG (C) mice or CB (CBMC in panel D) were compared for degranulation. Composite data of 3 (C) and 6 (D) experiments are shown. Numbers in plots represent frequencies within gates.

Figure 4

Figure 4

Preactivation enhances human NK-cell function in spleen cells from reconstituted NSG mice and human CB. Splenocytes from reconstituted NSG mice (A-B) purified NK cells from hu-NSG mice (C-D) and human CB (E-F) were used untreated (medium) or preactivated with poly I:C [p(I:C)] or the monokines IL-12 and IL-15. These cultures were restimulated with medium alone, monokines, K562 cells, or CEM cells. Degranulation (CD107a; A,C,E) and cytokine production (IFN-γ; B,D,F) were evaluated after gating on NKp46-positive cells. Panels A and B represent composite data from 4 mice in 2 experiments, panels C and D represent composite data from 10 mice in 2 experiments, and panels E and F represent composite data from 3 experiments.

Figure 5

Figure 5

Terminally differentiated CD16+ NK cells acquire functional capacity after IL-15–mediated preactivation. Distribution of KIR+ (A) and CD16+ (B) NK cells in splenic CD56bright, CD56dim, and CD56− NK-cell subsets of hu-NSG mice, as gated in the top panels of (A) in comparison to isotype control staining or (B) staining on marker negative CD3+ cells (gray shaded histograms). Degranulation (C) and IFN-γ production (D) of unstimulated and preactivated CD16+ and CD16− NK cells after coculture with K562 cells were analyzed. A representative experiment of 2 is shown in panels A and B, while panels C and D represent composite data of 2 independent experiments. Numbers in plots represent frequencies within gates or marker regions.

Figure 6

Figure 6

Cytotoxic ability of a human NK-cell line from reconstituted NSG mice. (A) Comparison of NKp46, KIR, perforin, and granzyme B (GrzB) expression of a reconstituted mouse-derived human NK-cell line with a line of human adult PB NK cells. (B) Cytotoxicity of the human NK-cell line from reconstituted NSG mice against K562. Loss of membrane integrity of PKH26-labeled K562 cells as a measure of cytotoxicity was assessed by To-Pro-3 iodide staining of DNA, which is blocked by intact cell membranes. Cytotoxicity was evaluated at the indicated NK:K562 (E:T) ratios. One representative of 6 experiments with 3 different NK-cell lines from 3 mice is shown. Numbers in plots represent frequencies within gates.

Figure 7

Figure 7

Preactivation of human NK cells of reconstituted NSG mice in vivo. Splenocytes of untreated or poly I:C [p(I:C)]-injected reconstituted NSG mice were restimulated ex vivo with medium alone or the monokines IL-12 and IL-15, or K562 cells, or CEM cells. Both degranulation (B) and cytokine production (IFN-γ; A,C) were evaluated. One representative of 2 experiments (A) and composite data (B) from 8 mice are shown. CFSE-labeled LCL721.221 (HLA class I negative) and LCL721.45 (HLA class I positive) cells were injected intravenously into untreated or poly I:C–preactiveted hu-NSG mice. Twelve hours later, the composition of CFSE-labeled cells in the recipient spleens was analyzed by w6/32 (anti–HLA class I) staining. Analysis of HLA class I expression and CFSE prior to adoptive transfer (D) and after recovery after gating on CFSE+ cells (E). One representative of 3 experiments is shown. Numbers in plots represent frequencies within gates or marker regions.

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