Chimeric NKG2D receptor-expressing T cells as an immunotherapy for multiple myeloma

Abstract

Objective: Most myeloma tumor cells from patients express NKG2D ligands. We have reported the development of a chimeric NKG2D receptor (chNKG2D), which consists of the NKG2D receptor fused to the CD3zeta chain. T cells expressing this receptor kill and produce cytokines in response to NKG2D-ligand+ tumor cells. Therefore, we investigated whether human chNKG2D T cells respond against human myeloma cells.

Materials and methods: ChNKG2D T cells were generated from healthy donors and myeloma patients. The effector phase of chNKG2D T cells was analyzed by cell-surface marker expression and human myeloma cell lines were tested for expression of NKG2D ligands. Lysis of myeloma cell lines and cytokine secretion by chNKG2D T cells was determined. ChNKG2D T cells grown in serum-free media, or cyropreserved, were assessed for effector cell functions.

Results: Myeloma cell lines expressed NKG2D ligands. ChNKG2D T cells from healthy donors and myeloma patients lysed myeloma cells, and secreted proinflammatory cytokines when cultured with myeloma cells or patient bone marrow, but not with peripheral blood mononuclear cells or normal bone marrow. Lysis of myeloma cells was dependent on chNKG2D T-cell expression of NKG2D and perforin. Additionally, chNKG2D T cells upregulated CD45RO, did not express CD57, and maintained expression of CD27, CD62L, and CCR7, indicating that the T cells were at an early effector stage. Finally, we showed that chNKG2D T cells generated with serum-free media, or when cryopreserved, maintained effector functions.

Conclusion: ChNKG2D T cells respond to human myeloma cells and can be generated using clinically applicable cell culture techniques.

Figure 1. Surface expression of NKG2D ligands on human myeloma and lymphoblastoid cell lines and differentiation markers on chNKG2D T cells

(A) Analyses using a soluble human NKG2D-mouseIgG1 fusion protein (filled histogram) or mouse IgG1 isotype control (open histogram) and a PE-anti-mouse IgG1 antisera was performed on RPMI8226, U266, MM.1s, or IM9 cells. (B) Ten days after stimulation with OKT3 and IL-2 and subsequent retroviral transduction, chNKG2D T cells from a healthy donor were analyzed for T cell differentiation markers (filled histograms) or isotype controls (open histograms). CD57, CD27, CD45 RO, CCR7, and CD62L expression are shown. Histograms are representative of data from two healthy donors.

Figure 2. ChNKG2D T cells lyse myeloma and lymphoblastoid cell lines in an NKG2D and perforin dependent manner

T cells from healthy donors were transduced with wtNKG2D (triangles) or chNKG2D (squares) and were used as effector cells in ⁵¹Cr release assays against (A) IM9, (B) MM.1s, (C,E) RPMI8226, or (D,F) U266 cells at the indicated E:T ratios (1:1, 5:1, or 25:1). ChNKG2D transduced T cells had higher specific lysis at all E:T ratios compared to wtNKG2D T cells, (p<0.05). (C and D) WtNKG2D and chNKG2D T cells were incubated with anti-NKG2D Abs (open symbols) or with isotype control IgG Abs (closed symbols) before addition to tumor cells. Blocking NKG2D significantly reduced the cytotoxicity of chNKG2D-transduced T cells against tumor cells at all E:T ratios compared with isotype control (P<0.05). (E and F) WtNKG2D and chNKG2D T cells were pre-treated with concanamycin A (CMA, open symbols) or EtOH vehicle (closed symbols) before addition of tumor cells. Inhibiting the perforin pathway by CMA significantly reduced the cytotoxicity of chNKG2D-transduced T cells against tumor cells at all E:T ratios compared with control (P<0.05). Data are represented as mean + SD of triplicates and are representative of 2 to 5 separate experiments.

Figure 3. ChNKG2D-transduced T cells from myeloma patients kill myeloma and lymphoblastoid cell lines

T cells from myeloma patients were transduced with wtNKG2D (open squares) or chNKG2D (closed squares) and used as effector cells in ⁵¹Cr release assays against (A) RPMI8226, (B) U266, (C) MM.1s, or (D) IM9 at the indicated E:T ratios (1:1, 5:1, 25:1). T cells from up to five different patients were tested on different days, with each line connecting the data points from a patient. ChNKG2D transduced T cells from myeloma patients had higher lysis compared to wtNKG2D T cells, (p<0.05). Data are represented as mean + SD.

Figure 4. Myeloma patient-derived chNKG2D T cells secrete proinflammatory cytokines and chemokines when cultured with myeloma cell lines

WtNKG2D-(open bars) and chNKG2D-transduced T cells (closed bars) from myeloma patients were cultured for 24 or 72 hours with RPMI 8226 cells, U266 cells, or media alone as indicated. Tumor cells were also cultured alone (hashed bars). Cell-free CM were analyzed for cytokines (IFNγ, GM-CSF, and TNFα,) and the chemokine CCL5. Data are presented as mean + SD and are representative of three different myeloma patient samples and one healthy donor. When cultured with tumor cells, chNKG2D T cells produced more cytokines than wtNKG2D T cells or tumor cells cultured alone (p<0.001).

Figure 5. ChNKG2D T cells secrete IFNγ when cultured with myeloma bone marrow but not with healthy PBMC or bone marrow cells

WtNKG2D (open bars) and chNKG2D T cells (closed bars) generated from healthy donors were cultured for 24 hours with (A) RPMI 8226 cells, U266 cells, autologous PBMCs (auto PBMC), healthy bone marrow (healthy BM), myeloma patient bone marrow cells (Pt A BM , Pt B BM), or media alone. (B) The wtNKG2D (open bars) and chNKG2D T cells (closed bars) were cultured with autologous PBMCs or healthy bone marrow spiked with 0.2%, 1%, or 5% RPMI8226 cells, as indicated. Tumor cells were also cultured alone (hashed bars). Cell-free CM was analyzed for IFNγ. Data are presented as mean + SD and are representative of T cells from two different healthy donors. An * indicates differences between wtNKG2D and chNKG2D samples (p<0.001).

Figure 6. ChNKG2D T cells cultured in serum-free media have similar anti-tumor efficacy as T cells grown in serum-containing media

(A) ChNKG2D T cells from a healthy donor were cultured in serum-containing media (RPMI-10%FCS,■) or serum-free media, either AIM V (♦), Stemline (▲), or X-Vivo 15 (X). The number of cells obtained with each media was counted over the course of the cell culture until the time of CD8⁺ T cell separation on day 8. (B) WtNKG2D (open squares) T cells and chNKG2D T cells (closed squares) cultured in RPMI-10% FCS and chNKG2D T cells cultured in Stemline medium (closed triangles) were used as effector cells in ⁵¹Cr release assays against RPMI8226 cells at the indicated E:T ratios (1:1, 5:1, or 25:1). ChNKG2D transduced T cells cultured in serum-containing and serum-free media had higher specific lysis at all E:T ratios compared to wtNKG2D T cells (p<0.05). (C) WtNKG2D (wt) and chNKG2D (ch) transduced T cells cultured in serum-containing media or serum-free media were cultured with myeloma cell lines for 24 hours, and cell-free CM were analyzed for IFNγ. Data are presented as mean + SD and are representative of 2 separate experiments. When cultured with tumor cells, chNKG2D T cells cultured in both serum-containing and serum-free media produced more IFNγ than wtNKG2D T cells (p<0.05).

Figure 7. Cryopreserved chNKG2D T cells maintain anti-tumor activity

(A) WtNKG2D (open symbols) and chNKG2D T cells (closed symbols) that were used fresh (squares) or that were cryopreserved and thawed one week later (triangles) were used as effector cells in ⁵¹Cr release assays against RPMI8226 cells at the indicated E:T ratios (1:1, 5:1, or 25:1). Both fresh and cryopreserved chNKG2D T cells had higher specific lysis at all E:T ratios compared to wtNKG2D T cells, (p<0.05). (B) Fresh and cryopreserved (frozen) wtNKG2D (wt) and chNKG2D (ch) T cells were cultured with myeloma cell lines for 24 hours, and cell-free CM were analyzed for IFNγ. Data are presented as mean + SD and are representative of 2 separate experiments. When cultured with tumor cells, fresh and cryopreserved chNKG2D T cells produced more IFNγ than wtNKG2D T cells (p<0.05).