Murine NK cell intrinsic cytokine-induced memory-like responses are maintained following homeostatic proliferation

Abstract

Several recent studies have demonstrated that innate immune NK cells exhibit memory-like properties with enhanced nonspecific and specific recall responses. Cytokine activation alone of murine NK cells induces the differentiation of memory-like cells that are more likely to produce IFN-γ, a key NK cell cytokine important for activation of the immune response. Using an adoptive cotransfer system, we first show that cytokine-induced memory-like responses are NK intrinsic. However, engraftment of donor NK cells in NK-competent hosts is poor because of homeostatic control mechanisms. Therefore, we used alymphoid Rag- and common γ-chain-deficient mice as recipients and observed homeostatic expansion of cotransferred cytokine-activated and control donor NK cells. Despite proliferation of all cells, NK cells derived from those cells originally activated by cytokines retained an intrinsic enhanced capacity to produce IFN-γ when restimulated in vitro with cytokines or target cells. These NK cell memory-like responses persisted for at least 4 wk in alymphoid hosts and 12 wk in NK-competent hosts. These findings indicate that memory-like NK cells can readily self-renew and maintain enhanced function in a lymphopenic host for at least a month.

Figure 1. Memory-like NK cell responses are cell-intrinsic

Congenic NK cells were enriched from Rag1^−/− mice and CFSE-labeled cytokine-activated and control cells adoptively transferred into Rag1^−/− hosts. (A) Host, control, and previously-activated (Pre-act) NK cells were identified by CFSE and/or CD45.2 as shown. Following re-stimulation for 4h with IL-12 + IL-15 significantly more pre-activated than control cells produce IFN-γ. Representative flow cytometry data from 7 day after adoptive transfer is shown, gated on NK1.1⁺ cells. (B) Splenic NK cells from recipient mice were harvested 1, 3, 7, and 21 days after adoptive transfer and assayed for IFN-γ production following culture with IL-12 + IL-15 (black bars) or media (gray bars) in vitro. Early after adoptive transfer, pre-activated NK cells were primed and produced very high levels of IFN-γ (Days 1 and 3). At later timepoints, pre-activated cells acquired a memory-like phenotype (Days 7 and 21) and were more likely to produce IFN-γ than control-transferred or host NK cells. (C) Donor pre-activated NK cells proliferate in vivo as shown by CFSE dilution (host NK cells shaded histogram; pre-activated NK cells solid line; control NK cells dashed line). (D) Most pre-activated NK cell (black bars) proliferation occurred within the first 3 days of transfer and very few control NK cells (open bars) proliferated in vivo (n.d., proliferation not detected). ****p<0.0001; ***p≤0.0001; **p≤0.001; *p<0.05. Graphed data represents mean +/− SEM of 3-7 independent experiments.

Figure 2. Homeostatic expansion of activated and control NK cells in alymphoid hosts

(A) Cytokine-activated or control treated congenic NK cells from Rag1^−/− mice were CFSE labeled and adoptively transferred into Rag-2^−/− γ_c^−/− hosts. Nearly all splenic NK cells proliferated within 7d of adoptive transfer as shown by dilution of CFSE of NK1.1⁺ pre-activated (solid line) and control (dashed line) NK cells and quantitated in panel B (mean +/− SEM of 4 independent experiments; n.s., not significant). (C) Percentage and absolute number of NK cells per spleen following adoptive transfer. (D) Ratio of pre-activated to control NK cells 1, 3, and 7 days after adoptive transfer.

Figure 3. Maintenance of a memory-like phenotype after NK cell expansion

Cytokine-activated or control treated congenic NK cells enriched from Rag1^−/− mice were adoptively transferred into Rag-2^−/− γ_c^−/− hosts. (A) Seven days after adoptive transfer, pre-activated NK cells were more likely to produce IFN-γ following a 4 hour in vitro stimulation with IL-12 + IL-15 as shown in a representative intracellular flow plot gating on NK1.1⁺ CD45.1⁺ control or CD45.1⁻ pre-activated NK cells. (B) Adoptively transferred cells were re-stimulated in vitro with IL-12 + IL-15 (black bars) or cultured with media alone (grey bars). Pre-activated NK cells had a primed phenotype with very high IFN-γ production 1d after adoptive transfer and spontaneous production of low levels of IFN-γ. By 7d, pre-activated NK cells exhibited a memory-like phenotype and were more likely to produce IFN-γ than control cells. Results represent the mean +/− SEM of 3-5 independent experiments. *p=0.01; **p=0.007.

Figure 4. Expanded memory-like NK cells production of IFN-γ in response to tumor targets and in vivo Poly(I:C)

(A & B) Cytokine-activated (Pre-act) and control-treated NK cells were adoptively transferred into separate Rag-2^−/− γ_c^−/− hosts. Transferred splenic NK cells were re-stimulated (A) 7d and (B) 4 weeks later in vitro with tumor targets (Yac-1 cells) alone or with IL-12. Significantly more memory-like (pre-act) than control cells produced IFN-γ at both timepoints. Data represent mean +/− SEM of 7 and 4 independent experiments, *p<0.05; **p<0.005). (C & D) Cytokine-activated and control-treated NK cells were adoptively co-transferred into Rag-2^−/− γ_c^−/− hosts and injected (C) 7d or (D) 4 weeks later with poly(I:C) and NK cell IFN-γ production measured (mean +/− SEM of 4 independent experiments, ****p<0.0001; no significant difference at 4 weeks).

Figure 5. Memory-like and control NK cells exhibit similar degranulation with tumor targets and expression of granzyme B

(A & B) Cytokine-activated (Pre-act, filled bars) and control-treated (white bars) NK cells were adoptively transferred into separate Rag-2^−/− γ_c^−/− hosts. Seven days after adoptive transfer, NK cell degranulation in response to co-culture with a tumor target (Yac-1 cells) was measured by assaying for cell surface CD107a. Pre-activated memory-like and control NK cells had similar degranulation with Yac-1 targets at (A) 1 and (B) 4 weeks after adoptive transfer. Results represent the mean +/− SEM of 3-4 independent experiments at each timepoint. (C) Intracellular expression of granzyme B was measured in pre-activated (solid line) and control NK cells (dotted line) 4 weeks after adoptive transfer co-transfer into Rag2^−/− γ_c^−/− hosts. Granzyme B expression of freshly isolated Rag-1^−/− NK cells is also shown (grey filled histogram). (D) Pre-activated and control NK cells expressed high levels of granzyme B (GzmB) 1 and 4 weeks after adoptive transfer. Results represent mean +/− SEM of 3 independent experiments at each timepoint, *p=0.047; n.s., not significant.

Figure 6. Long-term engraftment and production of IFN-γ by memory-like NK cells

Cytokine-activated (Pre-act) and control-treated NK cells were adoptively transferred into separate Rag-1^−/− hosts (A) or co-transferred into Rag-2^−/− γ_c^−/− hosts (B&C). NK cell IFN-γ responses following in vitro culture with IL-12 + IL-15 were measured 4 and 12 weeks later. (A) Following transfer into separate NK-competent hosts, pre-activated NK cells were more likely to produce IFN-γ than naïve host NK cells (*p=0.03 at 4 weeks and **p=0.007 at 12 weeks, mean +/− SEM of 3-5 independent experiments). There was no significant difference between the percentage of control-treated donor and host IFN-γ⁺ NK cells. (B) Following co-transfer of cells into alymphoid Rag-2^−/− γ_c^−/− hosts (as schematized in Figure 2, without CFSE labeling), the absolute number of splenic NK cells remained stable, while the ratio of pre-activated to control cells decreased between 1 and 12 weeks after adoptive transfer (mean +/− SEM of 3-8 mice per timepoint; data shown for 1 week is also shown in Figure 2C and D). (C) Four weeks after adoptive transfer, significantly more pre-activated than control NK cells produced IFN-γ. Twelve weeks after co-transfer into alymphoid hosts, slightly more control donor NK cells produce IFN-γ. (Mean +/− SEM of 4-5 independent experiments; black bars represent IL-12 + IL-15 stimulation, grey bars media; **p<0.004).