Characterizing the anti-tumor function of adoptively transferred NK cells in vivo

Abstract

Natural killer (NK) cells represent a promising cell type to utilize for effective adoptive immunotherapy. However, little is known about the important cytolytic molecules and signaling pathways used by NK cells in the adoptive transfer setting. To address this issue, we developed a novel mouse model to investigate the trafficking and mechanism of action of these cells. We demonstrate that methylcholanthrene-induced RKIK sarcoma cells were susceptible to NK cell-mediated lysis in vitro and in vivo following adoptive transfer of NK cells in C57BL/6 RAG-2(-/-)gammac(-/-) mice. Cytotoxic molecules perforin, granzymes B and M as well as the death ligand TRAIL and pro-inflammatory cytokine IFN-gamma were found to be important in the anti-tumor effect mediated by adoptively transferred NK cells. Importantly, we demonstrate that adoptively transferred NK cells could traffic to the tumor site and persisted in vivo which correlated with the anti-tumor effect observed. Overall, the results of this study have important implications for enhancing NK cell-based immunotherapies.

Fig. 1

Phenotypic characterization of the MCA-induced sarcoma cell line, RKIK-4862. Expression of TRAIL receptor DR5 (a), MHC Class I H2K^b (b), RAE1 molecules (c) and NKG2D ligands (d) was determined by staining with specific PE-conjugated antibodies or PE-conjugated NKG2D tetramer (solid lines). Isotype control-stained cells (dotted lines) were used as a control. Data shown are representative of three independent experiments

Fig. 2

Phenotypic characterization of IL-2-activated murine primary NK cells. The surface expression of various NK cell markers (a) NK1.1 (b) DX5 and (c) CD11b, activatory receptors (d) CD27, (e) TRAIL, (f) CD16/32, (g) NKG2D, activatory and inhibitory receptors (h NKG2A/C/E, i CD94, j KLRG1, k Ly49D, l 2B4), as well as activation status markers (m) CD69 and (n) B220, was analyzed by flow cytometry following staining with specific antibodies (solid line). Isotype control-stained NK cells were used as a control. Data shown are representative of three independent experiments

Fig. 3

In vitro sensitivity of RKIK tumor cells to NK cell-mediated lysis. Lysis of RKIK-4862 and RKIK-4654 tumor cells by IL-2-activated NK cells was compared to lysis of YAC-1 and RMA-S tumor cell lines using a ⁵¹Cr release assay. Lysis of RKIK-4862 sarcoma cells (closed triangles) by NK cells was equivalent to the killing of YAC-1 lymphoma cells (open squares), and higher than that observed for the RKIK-4654 sarcoma cell line (open triangles) and RMA-S tumor cells (closed squares). Data shown are average of triplicate wells ± SEM and are representative of two independent experiments

Fig. 4

Adoptively transferred NK cells can inhibit the growth of RKIK-4862 tumor in vivo. The ability of adoptively transferred NK cells to inhibit the growth of RKIK-4862 tumor cells was determined by s.c. injection of tumor cells followed by intravenous injection of 3 × 10⁶ IL-2-activated NK cells (days 0 and 1). Adoptively transferred NK cells were able to significantly inhibit the growth of s.c. RKIK-4862 tumor (open squares) compared to untreated tumor (closed squares, *p2 < 0.05). Results shown are average tumor area ± SEM from 16 and 12 mice for untreated and treated mice, respectively, from two independent experiments

Fig. 5

A critical role for perforin in the NK cell-mediated lysis of RKIK-4862 in vitro. The role of cytolytic molecules perforin, IFN-γ, granzymes (Gzm) A, B and M and death ligands TRAIL and FasL, in the NK cell-mediated lysis of RKIK-4862 cells was determined by isolating NK cells from mice deficient in these molecules. Lysis of RKIK-4862 tumor cells was determined using a ⁵¹Cr release assay. a WT NK (open squares) and IFN-γ^−/− NK cells (closed squares) were able to lyse RKIK-4862 at similar levels. In contrast, pfp^−/− NK cells (closed triangles) were unable to lyse these cells. b WT NK cells (open squares) and GzmA^−/− (closed squares), GzmB^−/− (closed triangles) or GzmM^−/− (open triangles) NK cells were able to equivalently lyse RKIK-4862 tumor cells. c WT NK cells (open squares), gld NK cells (closed triangles) and TRAIL^−/− NK cells (closed squares) were able to similarly lyse RKIK-4862 tumor cells. Results shown are average specific lysis ± SEM from triplicate wells and are representative of three independent experiments, *p2 < 0.05 and **p2 > 0.05

Fig. 6

Cytolytic effector molecules important for inhibition of RKIK-4862 tumor growth by adoptively transferred NK cells. To investigate the cytolytic effector molecules important for the inhibition of RKIK-4862 tumor growth, C57BL/6 RAG-2^−/−γc^−/− recipient mice bearing tumor were injected with NK cells derived from pfp^−/−, IFN-γ^−/−, TRAIL^−/−, GzmA^−/−, GzmB^−/−, GzmM^−/− or WT mice on days 0 and 1. To investigate the role of the NKG2D pathway, mice were treated with WT NK cells and intraperitoneal injections of the NKG2D blocking antibody C7 or isotype control antibody UC8.1B9. In all experiments adoptive transfer of WT NK cell-treated mice (open squares) significantly inhibited RKIK-4862 tumor growth compared to untreated mice (closed squares). a Adoptive transfer of pfp^−/− NK cells (open triangles) did not inhibit RKIK-4862 tumor growth compared to untreated mice (closed squares). b Adoptive transfer of GzmA^−/− NK cells (closed triangles) and WT NK cells (open squares) both led to significant inhibition of RKIK-4862 tumor growth compared to untreated mice (closed squares). c Adoptive transfer of GzmB^−/− NK cells (open triangles) did not inhibit RKIK-4862 tumor growth compared to untreated mice (closed squares). d Adoptive transfer of GzmM^−/− NK cells (open triangles) did not inhibit RKIK-4862 tumor growth compared to untreated mice (closed squares). e Adoptive transfer of TRAIL^−/− NK cells (open triangles) did not inhibit RKIK-4862 tumor growth compared to untreated mice (closed squares). f Adoptive transfer of WT NK cells with NKG2D blocking antibody C7 (closed triangles) or isotype control antibody UC8.1B9 (open triangles) led to significant inhibition of RKIK-4862 tumor growth compared to untreated mice (closed squares). g Adoptive transfer of IFN-γ^−/− NK cells (open triangles) did not inhibit RKIK-4862 tumor growth compared to untreated mice (closed squares). Data shown are average tumor area ± SEM from 5 to 15 mice from two independent experiments, **p2 > 0.05 and *p2 < 0.05

Fig. 7

Adoptively transferred NK cells persist in the peripheral blood of recipient mice. The persistence of adoptively transferred NK cells was investigated by transfer of congenic NK cells from C57BL/6-ptprc ^a mice (CD45.1⁺) into RKIK-4862 tumor-bearing C57BL/6 RAG-2^−/−γc^−/− mice (CD45.2⁺). Flow cytometry was used to detect CD45.1⁺ NK cells in the peripheral blood of NK cell-treated mice (closed bars) or non-treated mice (open bars). Adoptively transferred NK cells persist at detectable levels for at least 20 days post-transfer with respect to a the percentage of CD45.1⁺ circulating cells and b the number of CD45.1⁺ circulating cells per microliter of blood. Data shown are average ± SEM of six mice per time point, from two independent experiments

Fig. 8

Adoptively transferred NK cells can be detected in the liver, lungs, spleen and tumor of recipient mice. The presence of adoptively transferred NK cells in the spleens (gray bars), lungs (white bars) and livers (black bars) of mice receiving RKIK-4862 tumor and adoptively transferred, congenic CD45.1⁺ NK cells was investigated by flow cytometry. Adoptively transferred NK cells persist at detectable levels for at least 20 days post-transfer with respect to a the percentage of CD45.1⁺ cells in each organ and b the number of CD45.1⁺ circulating cells per milligram of organ. Data shown are average ± SEM from six mice from two independent experiments. c The presence of adoptively transferred NK cells in the tumor mass was investigated by flow cytometry to detect CD45.1⁺ NK cells following mechanical and enzymatic dissociation of the tumors from mice receiving RKIK-4862 tumor and adoptively transferred, congenic CD45.1⁺ NK cells (black bars) or RKIK-4862 alone (white bars) at day 17, post-transfer. Adoptively transferred NK cells could be detected in the tumor at this time point. p2 < 0.01 (NK cell-treated vs. non-treated groups). Data shown are average ± SEM from five mice per group