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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Gene Ther. 2011 Jan 6;18(5):509–516. doi: 10.1038/gt.2010.174

Treatment of multiple myeloma with adoptively transferred chimeric NKG2D receptor-expressing T cells

Amorette Barber 1, Kenneth R Meehan 2, Charles L Sentman 1
PMCID: PMC3095961  NIHMSID: NIHMS291460  PMID: 21209626

Abstract

Multiple myeloma causes approximately 10% of all hematologic malignancies. We have previously shown that human T cells expressing chimeric NKG2D receptors (chNKG2D) consisting of NKG2D fused to the CD3ζ cytoplasmic domain secrete proinflammatory cytokines and kill human myeloma cells. In this study, we show chNKG2D T cells are effective in a murine model of multiple myeloma. Mice with established 5T33MM-GFP tumors were treated with one or two infusions of chNKG2D T cells. Compared to mice treated with T cells expressing wtNKG2D, a single dose of chNKG2D T cells increased survival, with half of the chNKG2D T cell-treated mice surviving long-term. Two infusions of chNKG2D T cells led to tumor-free survival in all mice. ChNKG2D T cells were located at sites of tumor growth, including the bone marrow and spleen after i.v. injection. There was an increase in activated host T cells and NK cells at tumor sites and in serum IFNγ after chNKG2D T cell injection. Surviving mice were able to resist a rechallenge with 5T33MM cells but not RMA lymphoma cells, indicating that the mice developed a protective, specific memory response. These data demonstrate that chNKG2D T cells may be an effective adoptive cellular therapy for multiple myeloma.

Keywords: T cell, NK cell, chimeric receptor, cancer immunotherapy, myeloma

INTRODUCTION

Multiple myeloma, a B cell malignancy characterized by the proliferation of a plasma cell clone, causes approximately 20% of deaths due to hematologic malignancies in the United States. While current therapies including autologous stem cell transplant demonstrate high complete response rates, the majority of patients relapse1. As an alternative to standard treatments, immunotherapy may be able to specifically target tumor cells. Immunotherapeutic approaches used to treat myeloma include idiotype vaccination, dendritic cell therapies, and GM-CSF secreting tumor cell vaccines 2,3. Adoptive transfer of activated myeloma patient T cells or cells that are engineered to express TCRs specific for tumor-associated antigens is another promising therapeutic approach 2,4. A proportion of myeloma patients have circulating T cells that are specific for myeloma associated antigens, such as Mucin-1, NY-ESO-1, WT-1, or idiotype proteins, and the presence of tumor-specific T cells is associated with increased survival 2,4–9. However in patients with myeloma, the tumor cells have somehow escaped immune recognition. This could occur for many reasons, such as a lack of costimulation during T cell activation, down-regulation of MHC class I molecules, or the presence of suppressive factors, such as regulatory T cells or immunosuppressive cytokines. Treatments that target the tumor and activate the host immune system to target myeloma cells may be necessary for long-term benefit.

All human myeloma samples express ligands for the NKG2D receptor 10–13. Most healthy tissues do not express ligands for NKG2D, hence NKG2D ligands are potential tumor-specific targets 14,15. Human CD8+ T cells and NK cells are able to lyse myeloma cells and secrete proinflammatory cytokines after interactions with NKG2D ligands on myeloma cells 10,12,16–18. This indicates that targeting myeloma cells via the NKG2D receptor is a potential therapeutic opportunity. We have previously demonstrated that human T cells expressing chimeric NKG2D receptors (chNKG2D) consisting of NKG2D fused to the CD3ζ cytoplasmic domain lyse human myeloma cells 19. ChNKG2D T cells also secrete proinflammatory cytokines when cultured with primary myeloma bone marrow samples but not when cultured with healthy bone marrow cells or autologous PBMCs 19. In addition, treatment of tumor-bearing mice with chNKG2D T cells leads to long-term, tumor-free survival in murine models of ovarian cancer and lymphoma 20,21. This study determined the in vivo anti-tumor efficacy of chNKG2D T cells using a murine model of multiple myeloma and evaluated the induction of tumor-specific host immune responses.

RESULTS

ChNKG2D T cells lyse 5T33MM-GFP cells and produce IFNγ when cultured with murine myeloma cells

5T33MM is a myeloma cell line that formed spontaneously in an aging B6.Kalwrij mouse and was subsequently isolated and adapted to grow in cell culture 22,23. When injected intravenously into B6.Kalwrij mice, these tumor cells traffic to the bone marrow and spleen (but not the lymph nodes) and lead to a spike in serum immunoglobulin; thus, this model recapitulates features of human multiple myeloma in immunocompetent, syngeneic mice 24,25. This study investigated the potential of using chNKG2D T cells in vivo as a therapy for myeloma. To determine if 5T33MM cells were potential targets of chNKG2D T cells, the expression of NKG2D ligands was measured. 5T33MM-GFP cells expressed cell surface NKG2D ligands as detected by staining with a soluble NKG2D receptor (Figure 1A). RT-PCR for three murine NKG2D ligands was also performed, and 5T33MM-GFP cells expressed mRNA for Rae1 and Mult1 but not H-60 (Figure 1B). Rae-1 cell surface expression was also confirmed by staining with anti-Rae1 antibodies (data not shown). ChNKG2D T cells lysed 5T33MM-GFP cells, and this lysis was dependent on the NKG2D receptor because incubating the T cells with blocking anti-NKG2D antibodies prior to the assay abolished killing of the tumor cells (Figure 1C). ChNKG2D T cells also secreted significant amounts of IFNγ (23,730 +/− 798 pg/ml) compared to wtNKG2D T cells (6.4 +/− 5 pg/ml)when cultured with 5T33MM-GFP cells (Figure 1D). These data show that 5T33MM-GFP cells expressed NKG2D ligands and were recognized by chNKG2D T cells. These results were similar to previous data showing that human chNKG2D T cells lyse human myeloma cell lines in an NKG2D-dependent manner, and secrete proinflammatory cytokines, including IFNγ, when cultured with human myeloma cell lines and bone marrow from patients with myeloma 19.

Figure 1. ChNKG2D T cells lyse 5T33MM-GFP cells and produce of IFNγ when cultured with murine myeloma cells.

Figure 1

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(A) Staining with soluble mouse NKG2D-human IgG1 fusion protein (filled histogram) or human IgG1 isotype control (open histogram) was performed on 5T33MM-GFP cells. (B) cDNA isolated from 5T33MM-GFP cells was analyzed for NKG2D ligand expression by RT-PCR as follows: β-actin (lane 1) or mouse NKG2D ligands H-60 (lane 2), Mult-1 (lane 3), or Rae-1 (lane 4). (C) WtNKG2D (open squares) or chNKG2D (closed squares) T cells were used as effector cells with 51Cr labeled 5T33MM-GFP cells at the indicated E:T ratios (1:1, 5:1, 25:1). ChNKG2D T cells had higher specific lysis at all E:T ratios compared to wtNKG2D T cells (***- p<0.001). To show NKG2D dependence, wtNKG2D or chNKG2D T cells were incubated with anti-NKG2D Abs (dotted line), or with control IgG Abs (solid line) before incubation with tumor cells. Blocking NKG2D significantly reduced the cytotoxicity of chNKG2D-transduced T cells against tumor cells at all ratios compared with control (p<0.001). (D) wtNKG2D (open bars) or chNKG2D T cells (black bars) were cultured with irradiated 5T33MM-GFP cells or in media alone. After 24hr, chNKG2D T cells produced more IFNγ than wtNKG2D T cells when cultured with 5T33MM-GFP cells, (***- p<0.001). Data are represented as mean + SD and are representative of at least two experiments.

Treatment with chNKG2D T cells leads to long-term survival of 5T33MM-GFP bearing mice

To test the in vivo therapeutic efficacy of chNKG2D T cells against an established myeloma, 5T33MM-GFP cells were injected into B6.Kalwrij mice, and mice were given wtNKG2D or chNKG2D T cells. To determine whether mice had established tumors at the time of T cell treatment, the number of 5T33MM-GFP cells was measured in the spleen and bone marrow of tumor bearing mice beginning five days after tumor cell injection. Tumors were detected in the spleen and bone marrow five days after tumor cell injection, and increased over time (Figure 2A). Mice were given a single dose of wtN KG2D or chNKG2D T cells twelve days after tumor cell injection. The survival of the mice was measured. While wtNKG2D T cell-treated mice had a median survival of 28 days, all of the chNKG2D T cell treated mice had an increased survival, with approximately 50% of the chNKG2D T cell treated mice surviving long-term (five out of eleven mice) (Figure 2B). A previous study using a murine model of lymphoma had shown that multiple treatments with chNKG2D T cells enhanced anti-tumor efficacy 21. To increase the efficacy of treatment, tumor-bearing mice were injected with two treatments of wtNKG2D or chNKG2D T cells five and twelve days after tumor cell injection. All wtNKG2D T cell treated mice were sacrificed due to tumor growth while all of the chNKG2D T cell treated mice survived long-term (Figure 2C). These data show that chNKG2D T cell treatment of established myeloma increased survival, and multiple doses of chNKG2D T cells led to long-term survival in all mice.

Figure 2. Treatment with chNKG2D T cells leads to long term survival of 5T33MM–GFP bearing mice.

Figure 2

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5T33MM-GFP cells (3 × 105) were injected i.v. into B6.Kalwrij mice on day 0. (A) Mice were sacrificed five, twelve, sixteen, or twenty days after 5T33MM-GFP cell injection and tumor burden was determined by calculating the number of 5T33MM-GFP cells in the spleen and bone marrow (n=3). Naïve mice were also analyzed at each timepoint. Mice were treated i.v. with (B) a single treatment of 5 × 106 wtNKG2D (squares) or chNKG2D (diamonds) T cells after twelve days or (C) two doses of wtNKG2D T cells or chNKG2D T cells after five and twelve days. Survival of the mice was determined. Data are combined from two independent experiments. Significant differences are indicated (***-p < 0.001).

Lymphodepletion of the tumor-bearing host via radiation or chemotherapy has been shown to increase the in vivo efficacy of adoptively transferred T cells 26–30. To test whether lymphodepletion may increase the therapeutic potential of chNKG2D T cells, mice bearing 5T33MM-GFP tumor cells were injected with cyclophosphamide (180μg/g) or HBSS three days prior to injection of wtNKG2D or chNKG2D T cells. This cyclophosphamide treatment regimen is similar to what has been previously been shown to be lymphodepleting31,32. Pretreating mice with cyclophosphamide before chNKG2D T cell injection did not increase the survival of the mice any more than treatment with chNKG2D T cells alone. Two out of five chNKG2D T cell-treated mice survived long-term compared to two out of six mice treated with cyclophosphamide and chNKG2D T cells, indicating that lymphodepletion did not increase chNKG2D T cell anti-tumor efficacy (Figure 3A and B). To ensure that the treatment with cyclophosphamide was lymphodepleting, mice bearing 5T33MM-GFP tumor cells were injected with cyclophosphamide (180μg/g) or PBS, and the number of leukocytes was measured in the spleen and bone marrow after three days. As shown in Figure 3C, injection of cyclophosphamide led to a significant decrease in the number of CD19+ B cells, CD8+ and CD4+ T cells, NK cells, and GR1+ myeloid cells in the spleen and bone marrow of mice with 5T33MM-GFP tumor, compared to mice treated with PBS.

Figure 3. Treating mice with cyclophosphamide and chNKG2D T cells does not increase the efficacy of chNKG2D T cell treatment.

Figure 3

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5T33MM-GFP cells (3 × 105) were injected i.v. into B6.Kalwrij mice on day 0. Mice were treated i.p. with (A) 180μg/g of cyclophosphamide (cy, open symbols) on day 12 and treated i.v. with a single treatment of 5 × 106 wtNKG2D (squares) or chNKG2D (diamonds) T cells on day 15 or (B) 180μg/g of cyclophosphamide (open symbols) or HBSS (closed symbols) on day 9 and treated i.v. with a single treatment of HBSS (triangles) or 5 × 106 wtNKG2D (squares) or chNKG2D (diamonds) T cells on day 12. Survival of the mice was determined. Significant differences are indicated (*-p < 0.05). (C) 5T33MM-GFP cells (3 × 105) were injected i.v. on day 0 and mice were treated i.p. with PBS (open bars) or 180μg/g of cyclophosphamide (closed bars) on day 9. On day 12, mice were sacrificed and the number of CD19+, CD3+CD8+, CD3+CD4+, CD3−NK1.1+, and GR1+ cells in the spleen and bone marrow was determined. Data are shown as average +SD (n=4). Significant differences are indicated (*- p<0.001).

ChNKG2D T cells traffic to the bone marrow after i.v. injection in tumor bearing mice

5T33MM-GFP tumor cells were detected in the spleen and bone marrow, but not lymph nodes, after i.v. injection (data not shown) 33. The localization of chNKG2D T cells was studied to determine if the transferred cells are found in tumor-rich organs after i.v. injection. ChNKG2D T cells that expressed a congenic marker, Ly5.1, were injected i.v. into mice twelve days after 5T33MM-GFP cell injection, and the presence of Ly5.1+ cells in the bone marrow and spleen was determined. One day after T cell injection, chNKG2D T cells were found both in the spleen (14.4 × 104 chNKG2D T cells +/− 1.01 × 104 out of 9.31 × 107 total spleen cells) and in the bone marrow (2 × 104 chNKG2D T cells +/− 0.1 × 104 out of 1.24 × 107 total bone marrow cells), although at a lower percentage in the bone marrow (Figure 4). Nested PCR for the chNKG2D receptor was also used to detect chNKG2D T cells in the bone marrow and spleen, and one day after T cell injection, both the bone marrow and spleen were positive for chNKG2D T cells. Seven days after T cell injection, chNKG2D T cells were no longer detected in either tissue by flow cytometry. These data show that chNKG2D T cells were located in both the spleen and bone marrow, and that these T cells did not survive long-term in vivo.

Figure 4. ChNKG2D T cells are located in the bone marrow after i.v. injection in tumor bearing mice.

Figure 4

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(A) 5T33MM-GFP cells (3 × 105) were injected i.v. on day 0, and mice were treated with 5 × 106 chNKG2D T cells i.v. after twelve days. Spleen cells (black bars) and bone marrow cells (white bars) were isolated one and seven days after T cell injection, and the percent and number of Ly5.1+CD3+ cells was calculated. Data are shown as average +SD (n=4). (B) Nested PCR for the chNKG2D receptor was performed on DNA isolated from mice one day after T cell injection as follows: spleen cells from three wtNKG2D T cell-treated mice (lanes 1–3), spleen cells from three chNKG2D T cell-treated mice (lanes 4–6), spleen cells from a naïve mouse (lane 7), bone marrow cells from three wtNKG2D T cell-treated mice (lanes 8–10), bone marrow cells from three chNKG2D T cell-treated mice (lanes 11–13), bone marrow from a naïve mouse (lane 14), water (lane 15), 1 chNKG2D T cell (lane 16), 10 chNKG2D T cells (lane 17), 100 chNKG2D T cells (lane 18). Data are representative of two independent experiments.

ChNKG2D T cell treatment increases the activation of the host immune system

Previous studies in mouse models of ovarian cancer and lymphoma have shown that treatment with chNKG2D T cells induced a host immune response against tumor antigens and resulted in the mice demonstrating a resistance to tumor rechallenge 21,34. Activation of host cells was determined after chNKG2D T cell infusion. One day after chNKG2D T cell injection, the absolute numbers of host NK cells, CD4+, and CD8+ T cells did not change. However, host NK cells, CD4+, and CD8+ T cells in the bone marrow and spleen expressed more CD69 compared to mice treated with wtNKG2D T cells, indicating that host immune cells were more activated after chNKG2D T cell treatment (Figure 5). Therefore, not only do chNKG2D T cells have direct anti-tumor effects on 5T33MM-GFP cells, but chNKG2D T cells contribute to the activation of host immune cells which may target other tumor antigens.

Figure 5. ChNKG2D T cell treatment induces activation of the host immune response.

Figure 5

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5T33MM-GFP cells (3 × 105) were injected i.v. on day 0, and mice were treated with 5 × 106 wtNKG2D (white bars) or chNKG2D (black bars) T cells i.v. after twelve days. Bone marrow and spleen cells were isolated one day after T cell injection and (A) the number of CD3−NK1.1+, Ly5.1−CD4+, and Ly5.1−CD8+ cells or (B) the number of CD3−NK1.1+CD69+, Ly5.1−CD4+CD69+, and Ly5.1−CD8+CD69+ cells was determined. Data are shown as average +SD (n=4) and are representative of two experiments. (C) One and seven days after injection of wtNKG2D (white bars) or chNKG2D (black bars) T cells, amount of IFNγ in the serum was analyzed. Serum from naïve mice (hashed) was also analyzed (<80 pg/ml). Data are presented as average + SD (n=6–8) and are combined from three independent experiments. Significant differences are indicated (* -p < 0.05, ***- p<0.001).

We have previously shown in murine models of lymphoma and ovarian cancer that one effector molecule required for chNKG2D T cell anti-tumor efficacy is IFNγ 21,34–36. To determine if IFNγ was increased in vivo after chNKG2D T cell treatment in 5T33MM-GFP tumor-bearing mice, the amount of IFNγ in the serum of tumor-bearing mice was measured. Serum IFNγ was increased in tumor-bearing mice treated with chNKG2D T cells compared to wtNKG2D T cell treated mice (Figure 5C). The increase in serum IFNγ was sustained for at least one week after chNKG2D T cell injection, even though the chNKG2D T cells were no longer present in significant numbers. Together these data show that chNKG2D T cell treatment led to an increase in the activation of the host immune cells and in systemic IFNγ levels, indicating that chNKG2D T cell treatment induced a host immune response to the tumor.

Tumor-surviving mice develop protective host memory responses to 5T33MM tumor antigens

To test whether tumor-surviving mice developed protective memory responses to tumor antigens, spleen cells from naïve mice or chNKG2D T cell treated surviving mice from figure 2B were cultured with media only, 5T33MM cells, or with tumor cells the mice had not previously rejected, RMA and RMA-Rae1 cells, both murine T cell lymphomas. Spleen cells from tumor surviving mice secreted significant amounts of IFNγ when cultured with 5T33MM cells, but did not secrete IFNγ when cultured with media alone or with tumor cells the mice had not previously encountered, even if these tumor cells expressed ligands for NKG2D (Figure 6A). Spleen cells from naïve mice did not secrete IFNγ in any of the culture conditions. To determine which cells were producing IFNγ, intracellular staining was performed. As shown in Figure 6B, both CD8+ and CD4+ T cells from tumor-surviving mice produced IFNγ when cultured with 5T33MM cells, but not when cultured in media alone or with RMA-Rae1 cells. This showed that chNKG2D T cell treated tumor-surviving mice developed host memory responses to 5T33MM tumor antigens.

Figure 6. ChNKG2D T cell treated tumor-surviving mice develop protective immunity to 5T33MM tumor antigens.

Figure 6

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Eighty days after tumor injection, spleen cells from chNKG2D T cell treated from figure 2B (n=5) and naive B6.Kalwrij mice (n=4) were cultured with RMA cells (open bars), RMA-Rae1 cells (grey bars), 5T33MM cells (filled bars), or media alone (hatched bars). (A) Cell-free supernatants were analyzed for IFNγ after 72 hours or (B) intracellular cytokine staining was performed after 24 hours for IFNγ production and gated on CD8+ or CD4+ T cells as indicated. Cells from tumor surviving mice produced significantly more IFNγ when cultured with 5T33MM cells than naïve mice, as indicated. Significant differences are indicated (***-p < 0.001). Data are represented as mean + SD. (C) 5T33MM-luciferase (5T33MM-Luc, triangles) or RMA-Rae1-Luciferase cells (RMA-RL, squares) (3 × 105) were injected i.v. into surviving mice 80 days after original tumor challenge (open symbols, from figure 2C) or naïve B6.Kalwrij mice (closed symbols). Survival of the mice was determined. Data are combined from two independent experiments. Significant differences are indicated (***-p < 0.001).

To determine if the host memory responses were protective against a specific tumor rechallenge, 5T33MM-Luciferase cells were injected i.v. into tumor-surviving mice from Figure 2C, 80 days after original tumor injection. Tumor cells were also injected into naïve mice as a tumor growth control. All of the tumor-surviving mice rejected the tumor rechallenge with 5T33MM-Luciferase cells, whereas 5T33MM-Luciferase bearing naïve mice had a median survival of 27 days (Figure 6C). RMA-Rae-Luciferase cells, a tumor cell line these mice had not previously been exposed to, were injected i.v. into additional 5T33MM-GFP tumor-surviving mice. RMA-Rae1-Luciferase cells grew at a similar rate in tumor-surviving mice and naïve mice, indicating that the protective memory response was specific for 5T33MM tumor cell antigens.

DISCUSSION

These data show that treatment with chNKG2D T cells leads to long-term survival of mice bearing established multiple myeloma. In addition to increasing the survival of the mice, chNKG2D T cells are located in the bone marrow and increase the activation of host immune cells at the tumor site. Tumor surviving mice develop protective host immune responses that are specific to myeloma cell antigens because surviving mice were able to reject a tumor rechallenge with myeloma cells but were unable to reject lymphoma cells. These findings combined with previous data in other tumor models indicate that chNKG2D T cell therapy can lead to epitope spreading and the induction of long-term protective immunity.

Several mouse models for myeloma have been developed, with the SCID-hu model and the 5T murine models being two of the most frequently used models 37. One benefit of using a SCID-hu model is that this model uses primary human myeloma cells inoculated into SCID mice38. However engrafting human cells into mice requires the use of immunodeficient mice, therefore this model is not optimal for evaluating therapeutic approaches that induce activation of the host immune system. Additionally, potential xenogeneic responses of infused human effector cells (i.e. human anti-mouse responses) may occur complicating data interpretation. An ideal mouse model for immunotherapeutic studies uses syngeneic tumor cells in an immunocompetent host. The 5T33MM murine myeloma model fulfills these criteria as injection of 5T33MM cells into B6.Kalwrij hosts leads to myeloma formation with symptoms similar to human myeloma 37. Use of the 5T33MM mouse model not only allowed for the investigation of chNKG2D T cell efficacy against myeloma cells in vivo, but also for the study of the induction of host immune responses to tumor antigens that would not be possible if an immunodeficient mouse model had been used. In addition, the 5T33MM murine model elicits a similar immunosuppressive environment as the human disease, including an increase in regulatory T cells, myeloid derived suppressor cells, and TGFβ 39,40. Thus this model also allows the study of the efficacy of chNKG2D T cells in an immunosuppressive environment that T cells would likely encounter in patients.

The role of different immune cells in developing productive anti-tumor responses against myeloma remains unclear. There is evidence that both CD8+ T cells and NK cells are able to lyse myeloma cells and to reduce myeloma tumor burden after activation with antigen or cytokines 17,41,42. The presence of tumor antigen-specific T cells in myeloma patients correlates with improved prognosis, and patient CD8+ T cells can lyse myeloma cells after activation, with lysis being dependent on MHC class I and perforin expression 2,4,6,7,43. However, there is much evidence that myeloma patients are immunosuppressed, which is partially due to myeloma cell expression of FasL, IL-10, IL-6, VEGF, Muc-1, and TGFβ 44. Increased amounts of these molecules, along with an increased number of regulatory T cells, can inhibit immune responses in myeloma patients 45–47. Thus therapies that are able to reduce the effect of immunosuppressive mechanisms and fully activate the host immune response to respond to the tumor cells may demonstrate improved outcomes. In this study, chNKG2D T cell treatment not only lead to tumor-free survival, but also the activation of the host immune response, as seen by an increase in activated host NK cells and T cells at the tumor site, and an increase in IFNγ in the serum of the mice. This host immune response was also protective against a tumor rechallenge. These data indicate that chNKG2D T cell therapy is a novel way to activate the host immune system to respond to myeloma antigens and may decrease tumor relapse by developing protective memory responses.

Unlike other adoptive cell therapy approaches that utilize tumor-specific T cells, treatment with chNKG2D T cells was not enhanced by lymphodepletion of the host. Proposed benefits of depleting immune cells prior to T cell transfer include 1) long-term survival of transferred T cells through decreased competition for antigen presenting cells and homeostatic cytokines, 2) decreasing immunosuppressive cells, such as myeloid derived suppressor cells and regulatory T cells, which decrease the anti-tumor responses of transferred T cells, 3) induction of homeostatic proliferation and activation of host T cells, and 4) expansion of immature dendritic cells that can present tumor antigens 27,32,48–51. While lymphodepletion has been shown to increase the survival of transferred T cells, this study shows that although chNKG2D T cells do not survive long-term, they instead induce long-lived anti-tumor immune responses in the host. It has been previously demonstrated that host immune cells are essential for anti-tumor efficacy of chNKG2D T cells 35. The presence of host leukocytes at the time of chNKG2D T cell injection is likely important for the induction of host immune responses, thus lymphodepletion may inhibit the development of the host anti-tumor response that results from chNKG2D T cell treatment. Additionally, previous data have shown that chNKG2D T cells are able to decrease the number of regulatory T cells at the tumor site and activate host myeloid cells so that the local myeloid cells switch from an immunosuppressive to an immunostimulatory phenotype 36. Since treatment with chNKG2D T cells recapitulated many of the benefits of lymphodepletion, including reducing immunosuppression and activating host immune cells, lymphodepleting the host prior to chNKG2D T cell treatment does not appear necessary or beneficial.

Current chemotherapies for myeloma are inadequate. While treatment with these drugs or autologous transplant offers a high response rate, the majority of patients still relapse. NKG2D ligand expression is partially regulated by the induction of the ATM/ATR DNA damage pathway and by ubiquitination and subsequent proteasomal degradation, thus chemotherapeuic agents that induce DNA damage or inhibit the proteasome may increase NKG2D ligand expression on the surface of cells 52,53. Supporting this idea, many agents used to treat myeloma, including bortezomib, melphalan, and doxorubicin, increase NKG2D ligand expression on primary myeloma cells, while thalidomide and dexamethosone did not 13,18,54. It is possible that coupling treatment with chemotherapeutic agents that upregulate the expression of NKG2D ligands with chNKG2D T cell therapy may further enhance the therapeutic efficacy of chNKG2D T cells. However additional studies should be performed to determine if these compounds also upregulate NKG2D ligands on normal cells, which would might result in recognition of healthy tissues by chNKG2D T cells.

Treatment with chNKG2D T cells is a novel approach for treating myeloma. Treatment with chNKG2D T cells increased survival in mice bearing an established 5T33MM-GFP tumor, and these mice also developed protective host immune responses to tumor antigens. Unlike other adoptive cell therapy approaches, this was not enhanced by lymphodepletion of the host. All long-term surviving mice appeared healthy in this study and in previous studies, even 300 days after chNKG2D T cell injection, indicating that treatment of tumor-bearing mice with chNKG2D T cells did not cause significant side effects 21,34. The fact that chNKG2D T cells do not survive long-term to reconstitute the host may be a beneficial attribute and will minimize potential unwanted side effects. These data suggest that chNKG2D T cells are a potentially useful and novel immunotherapeutic approach for multiple myeloma.

MATERIALS AND METHODS

Mice

C57Bl/6 Ly5.2+ mice were purchased from the National Cancer Institute (Frederick, MD). C57Bl/Kalwrij mice were kindly provided by Dr. Randolph Noelle (Dartmouth Medical School, Lebanon, NH). Male and female mice were between 7 and 10 weeks of age at the start of the experiments. All animal work was performed in the Dartmouth Medical School Animal Facility in accordance with Institutional guidelines.

Treatment of mice with 5T33MM cells and genetically modified T cells

Mouse spleen cells were isolated and transduced with chNKG2D or wtNKG2D genes as previously described 20,55. 5T33MM-GFP and 5T33MM-Luciferase cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM pyruvate, 10 mM Hepes, 0.1 mM non-essential amino acids and 50 μM 2-mercaptoethanol. 5T33MM-GFP cells (3 × 105) were injected intravenously (i.v.) into C57Bl/Kalwrij mice. For survival experiments, two doses of 5 × 106 wtNKG2D or chNKG2D T cells were transferred five and twelve days after tumor cell injection or a single dose of 5 × 106 wtNKG2D or chNKG2D T cells was transferred twelve days after tumor cell injection. Mice were monitored daily and were sacrificed when they showed signs of morbidity, such as dragging of hind legs, scruffiness, or labored breathing. For tumor rechallenge experiments, 3 × 105 5T33MM-Luciferase or 3 × 105 RMA-Rae1-Luciferase cells were injected i.v. into naïve mice or tumor-surviving mice (day +80) that had previously received two doses of chNKG2D T cells on days five and twelve and survival of the mice was measured. In some experiments, cyclophosphamide (180μg/g, Sigma) was administered intraperitoneally three days prior to T cell injection.

Cytokine production and cytotoxicity by chNKG2D T cells

WtNKG2D or chNKG2D T cells (106) were cultured with 5T33MM-GFP cells (106) or in media alone. Cell-free conditioned media were collected after 24 hours and assayed for IFNγ using mouse Duoset ELISA kits (R&D Systems). Lysis of 5T33MM-GFP cells was determined by 51Cr release assays, as previously described 20,55. To block NKG2D receptors, T cells were preincubated at 37°C for two hours with anti-NKG2D mAbs (clone CX5, 20μg/ml, sodium azide free) or isotype control mAbs prior to addition of target cells.

RT-PCR and nested PCR

Total RNA from 5T33MM-GFP cells was extracted and used with primers for amplification of mouse NKG2D ligands, as previously described 20. For nested PCR, total DNA was isolated from spleen cells and bone marrow cells using a lysis buffer (50 mM Tris-HCl, 100mM NaCl, 0.1% Triton-X100, 1mg/ml proteinase K) at 5 × 106 cells/30μl. Cells were lysed at 55°C overnight, and proteinase K was inactivated at 98°C for 10 minutes. These samples were directly used as template for nested PCR. The primers for the pFBneo vector and chNKG2D were used as previously described 34. PCR products were run on agarose gels and visualized by staining with SYBR Safe (Invitrogen).

Flow cytometry

For FACS analysis of NKG2D ligand expression, 5T33MM-GFP cells were stained with a mouse NKG2D-human IgG1 fusion protein (R&D Systems) and an APC-labeled goat anti-human IgG secondary (Jackson ImmunoResearch, Westgrove, PA) or with APC-anti-Rae1 (186107, R&D Systems). For 5T33MM-GFP tumor burden analysis, mice were sacrificed five, twelve, sixteen, or twenty days after tumor cell injection and analyzed for percent and absolute number of GFP+ cells in the spleen and bone marrow. For analysis of T cell localization and host cell activation, mice were sacrificed one or seven days after T cell injection. Spleen and bone marrow cells were incubated with FcR block and mouse γ globulin (Jackson ImmunoResearch) to prevent non-specific binding, and stained with FITC-conjugated anti-CD3 (clone 145-2C11) or anti-CD4 (clone GK1.5), PE-conjugated anti-CD3, APC-conjugated anti-CD45.1 (clone A20) or anti-NK1.1 (clone PK136), and biotin-conjugated anti-CD69 (clone H1.2F3) with a secondary incubation with PE-Cy5.5 conjugated streptavidin. For analysis of lymphodepletion by cyclophosphamide, mice were sacrificed three days after cyclophosphamide treatment. Spleen and bone marrow cells were incubated with FcR block and mouse γ globulin, and stained with PE-conjugated anti-CD3 (clone 145-2C11) or anti-CD19 (clone 6D5), APC-conjugated anti-CD8 (clone H35-17.2) or anti-GR1 (clone RB6-8C5), and biotin-conjugated anti-CD3 (clone 145-2C11) or anti-NK1.1 (clone PK136) with a secondary incubation with PE-Cy5.5 conjugated streptavidin. All isotype controls were obtained from eBiosciences (San Diego, CA). Cell fluorescence was monitored using a FACSCalibur cytometer (Becton Dickinson, San Jose, CA) or an Accuri C6 cytometer (Accuri Cytometers Inc, Ann Arbor, MI).

Cytokine production in secondary stimulation cultures and in serum

Spleen cells (2.5 ×106) from naïve or day 80 tumor-surviving mice were cultured with irradiated (120 Gys) 5T33MM, RMA-Rae1, or RMA cells (2.5 × 105) for 72 hours. Cell free conditioned media or serum samples taken directly from mice were assayed for IFNγ by ELISA using mouse Duoset ELISA kits (R&D Systems). For intracellular staining, 10μg/ml brefeldin A was added to the wells (Sigma) during the last five hours of culture. Cells were then incubated with FcR block and cell-surface stained with FITC-conjugated anti-CD8β (clone CT-CD8β, ebioscience) and APC-conjugated anti-CD4 (clone RM4-5, BD Biosciences). Cells were fixed with 1% paraformaldehyde, permeabilized with 0.1% saponin, and stained with PE-conjugated anti-IFNγ (clone XMG12, ebioscience) or PE-conjugated anti-rat IgG1 isotype control (ebioscience).

Statistical analysis

Differences between groups were analyzed using the Student’s t-test or ANOVA, as appropriate. Values of p<0.05 were considered significant. For survival studies, Kaplan-Meier survival curves were plotted and analyzed using the Log rank test and Prism software (GraphPad Software, San Diego, CA).

Acknowledgments

The authors wish to thank Karin Vanderkerken for the 5T33MM tumor cells, Gary Ward and Alice Givan at the Englert Cell Analysis Laboratory for assistance with flow cytometry (Norris Cotton Cancer Center, Lebanon, NH), and the Animal Resource Center at Dartmouth Medical School for help with the animal studies. The authors also thank Randy Noelle for helpful comments on the study design and manuscript.

This work was supported in part by grants from the Norris Cotton Cancer Center at Dartmouth Medical School, Department of Microbiology and Immunology, and National Institutes of Health (CA130911, T32 AI07363, P20 RR16437 (KRM)). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Footnotes

CONFLICT OF INTEREST

The technology described in this paper is licensed by Celdara Medical, LLC. Dr. Sentman and Celdara are developing the technology for clinical use. If they are successful, Dr. Sentman will receive compensation. This arrangement is under compliance with the policies of Dartmouth College.

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