Purpose: To explore the potential of ex vivo expanded healthy donor–derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients.
Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry–based in vitro killing assays and xenograft models against AML patient blasts and healthy donor–derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays.
Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor–unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands’ expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1–blocking antibodies.
Conclusions: This study supports healthy donor–derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370–82. ©2017 AACR.
This article is featured in Highlights of This Issue, p. 251
Chemotherapy resistance represents a significant barrier to acute myeloid leukemia (AML) therapy. Different forms of cellular therapy have been developed to overcome this barrier, but low efficacy and associated toxicity have hampered their wide use in clinic. Here, we demonstrate that double-negative T cells (DNT) from healthy individuals can be expanded ex vivo to therapeutic levels under GMP conditions and be cryopreserved. Expanded human DNTs target a large array of primary AML cells, including chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor–unrestricted manner. Importantly, allogeneic DNTs neither attack normal human cells nor cause xenogeneic GVHD. Collectively, healthy donor–derived allogeneic DNTs provide a potential new off-the-shelf cellular therapy that is safe and effective to treat patients with chemotherapy-resistant AML. A first-in-human phase I clinical trial (NCT03027102) using allogeneic DNTs to treat patients with high-risk AML has been initiated.
Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with 5-year survival rates of approximately 5% and 35% for elderly and younger patients, respectively (1–4). Despite decades of using chemotherapy to treat AML patients, a high relapse rate and refractoriness to chemotherapy are major challenges to patient survival (1–3). Allogeneic hematopoietic stem cell transplantation is a potential curative cellular therapy for chemotherapy-resistant leukemia (5–7), but limited donor availability, the risk of GVHD, and other transplantation-related toxicities restrict its wide application in elderly and debilitated patients (4, 7–10).
Recently, chimeric antigen receptor (CAR)-T-cell therapies have shown excellent clinical responses against lymphocytic leukemia (11–13) and promising results in preclinical AML studies (14–17). However, in clinical trials, CAR-T cells specific for CD33 and LeY antigen caused severe toxicity in some of the treated AML patients (18, 19). Also, genetic modification of infused cells increases the treatment cost and potential risk to patients (17). Multiple clinical trials using allogeneic natural killer (NK) cell therapy to treat AML patients demonstrated the safety and clinical benefits, but a noticeable number of patients either did not achieve complete remission or experienced disease relapse (20–25). Some studies highlighted the importance of precise HLA-KIR matching for improved efficacy of NK therapy (24, 25), but this can limit treatment availability to patients. Furthermore, long-term efficacy of NK-cell therapy has yet to be demonstrated. Therefore, the need for safer and more effective cellular therapies with a broader and easier clinical applicability to target chemotherapy-resistant AML and prevent disease relapse remains.
In this study, we explored a nonconventional T-cell subset named double-negative T cells (DNT) as a novel therapy to target AML, including leukemic cells that are resistant to the conventional chemotherapy. DNTs are mature peripheral T lymphocytes expressing CD3–T-cell receptor (TCR) complex but not CD4, CD8, nor invariant NK T-cell markers. They represent 1% to 3% of peripheral blood mononuclear cells (PBMC; ref. 26). The function of human DNTs was largely unknown due to their low frequency in vivo and a lack of an effective method to expand them to sufficient numbers for in vivo studies. We have shown previously that DNTs expanded from AML patients were cytotoxic to autologous AML cells in vitro (26). However, attempts to expand autologous DNTs for treating AML patients have failed perhaps due to the defect of DNTs in patients. Whether allogeneic DNTs expanded from healthy individuals can effectively target AML cells in vitro and in vivo while sparing nonmalignant cells and tissues of recipients, and the mechanisms involved, has not been explored previously.
Here, we demonstrate, for the first time, that therapeutic quality and quantity of DNTs can be expanded ex vivo from healthy donors (HD) and that these cells can selectively target a large array of primary AML samples, including those from chemotherapy-resistant patients without observed toxicity toward normal cells and tissues. Furthermore, we identified a positive feedback loop of NKG2D, DNAM-1, and IFNγ, which helps to explain the ability of DNTs to selectively recognize and target AML but not normal cells. Collectively, our findings open a new window of cellular therapy using DNTs expanded from healthy volunteers as a potential off-the-shelf product to treat patients with high-risk AML, and perhaps other cancers.
Materials and Methods
DNTs and leukemic cell lines
DNTs were expanded ex vivo as described previously (26). Briefly, DNTs enriched by depleting CD4+ and CD8+ cells from PBMCs using CD4− and CD8− depletion cocktail (Stemcell Technologies) were cultured in anti-CD3 antibody-coated plates (OKT3; 5 μg/mL) for 3 days in RPMI1640 supplemented with 10% FBS and 250 IU/mL of IL2 (Proleukin, Novartis Pharmaceuticals); soluble anti-CD3 (0.1 μg/mL) was added on day 7, 10, and 14. On days 3, 7, and 10, fresh media and IL2 were added. DNTs were harvested 10 to 20 days postexpansion for subsequent experiments. The leukemic cell lines AML3/OCI (AML3), KG1a, and MV4-11 were obtained from ATCC.
Flow cytometry–based in vitro killing assay
DNTs stained with PKH-26 (Sigma) were cocultured with target cells for 2 to 4 hours; cells were then stained with anti-human CD3 (HIT3a), CD33 (WM53), CD45 (HI30) FITC-Annexin V, and 7AAD (all from BioLegend) and analyzed using flow cytometry. Specific killing was calculated by: . For blocking assays, DNTs were incubated with neutralizing antibodies for 0.5 to 1 hour prior to coincubation with target cells at 4:1 effector-to-target ratio for 2 hours or 2:1 effector-to-target ratio for 4 hours. Percent inhibition of killing was calculated by . For IFNγ pretreatment assays, DNTs or AML cells were pretreated with 50 ng/mL of recombinant human IFNγ (BioLegend) for 1 hour or overnight. Percent increase in killing was calculated by .
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory) were maintained at UHN animal facility. Eight- to 12-week-old females were irradiated (250 cGy) 24 hours prior to intrafemoral or tail vein injection of the 2–5 × 106 primary AML blasts. DNTs (2 × 107) were injected intravenously at the indicated time points. rIL2 (Proleukin) was administered (104 IU/mouse) intraperitoneally concordant with the DNT injections on days 1, 2, 4, and 7 and weekly thereafter until euthanized. Two to 4 weeks after the last DNT injection, mice were sacrificed and spleen and bone marrow cells were harvested, and the frequency of AML was analyzed by flow cytometry. For GVHD-related experiments, 5–20 × 107 DNT, PBS, or 5 × 106 PBMCs were intravenously injected into irradiated naïve NSG mice, and percent body weight change was calculated as . Mice with weight loss greater than 25% were euthanized via cervical dislocation. Fourteen days postinjection, liver, lung, and small intestine were obtained, fixed in 10% formalin for 24 to 48 hours, and then in 70% ethanol until samples were sent for hematoxylin and eosin histologic analysis. Histologic staining and picture acquisition was done by the Advanced Optical Microscopy Facility at UHN. For safety-related experiments, irradiated NSG mice were injected with 3 × 105 CD34+CD133+ hematopoietic stem/progenitor cells (HSPC) before treated with DNTs.
All graphs and statistical analysis were generated using GraphPad Prism 5. Student t test and linear regression test were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 indicate significance between experimental and control values. Error bars represent ±SEM.
Human samples and study approval
Human blood, bone marrow, and CD34+ cells were collected from healthy adult donors and AML patients after obtaining written informed consent and used according to University Health Network (UHN) Research Ethics Board (05-0221-T) and NHLBI-approved protocols. PBMCs from HD or AML patients were separated by Ficoll (GE Healthcare) density gradient. AML patient samples were viably frozen in the Princess Margaret Leukemia Bank and stored in liquid nitrogen until used. Animal studies were approved by the Institutional Animal Care Committee of the UHN (permit number: 741.22) and carried out in accordance with the Canadian Council on Animal Care Guidelines.
Antileukemic function of HD-derived DNTs
To explore the potential of using allogeneic human DNTs to treat leukemia, we developed a protocol allowing for a large-scale GMP expansion of DNTs from HDs. So far, we have expanded DNTs from more than 60 HDs, of which 6 expansions were done under GMP conditions. On average, 1.41 ± 0.51 × 108 DNTs were obtained from each milliliter of peripheral blood after 17 to 20 days ex vivo expansion with >90% purity (Fig. 1A). The ex vivo expanded DNTs expressed CD3 and αβ- or δγ- TCR but not CD4, CD8, or iNKT cell markers (Fig. 1B) and exhibited a central/effector memory T-cell phenotype (Supplementary Fig. S1). As αβ-TCR+ and δγ-TCR+ DNT subsets display comparable antileukemic function (26), for the simplicity of clinical applications, no further selection for specific TCRs was performed for the subsequent studies when more than 90% of cells in expanded products were DNTs.
Expanded DNTs effectively killed 36 of 46 primary AML patient samples (patient information shown in Supplementary Table S1; Fig. 1C) in a dose-dependent manner (Supplementary Fig. S2), albeit with heterogeneity in the level of cytotoxicity to each patient sample. Higher effector-to-target ratio (Supplementary Fig. S2A) or longer incubation time (Supplementary Fig. S2B) showed increased killing. The threshold of 10% was used to distinguish susceptible and resistant AML samples, as samples with specific killing greater than 10% showed a dose-dependent cytotoxicity, whereas those with lower than 10% killing did not (Supplementary Fig. S2C). No correlation was observed between the susceptibility to DNTs and the patient's age at diagnosis, WBC count, or MRC cytogenetic risk groups (Supplementary Fig. S3A–S3C). However, significantly lower cytolysis was observed in samples obtained from patients with lower percentages of AML cells in the bone marrow, in male patients, and patients with AML secondary to prior myelodysplastic syndrome (Supplementary Fig. S3D–S3F). On the other hand, samples obtained from M5 FAB classified AML patients showed a higher level of susceptibility to DNT-mediated cytotoxicity than the rest of the patient samples tested (Supplementary Fig. S3G).
To determine the relative potency of DNTs in comparison with other cytotoxic cells, DNTs from two HDs and NK92, an NK-cell line used in a clinical trial for AML patient treatment (NCT00900809; ref. 27), were used as effectors against eight leukemia targets. Although K562, a natural NK-cell target, was effectively killed at a comparable level by both DNTs and NK92, all six primary AML blasts and an AML3/OCI cell line were more susceptible to DNT- than NK92-mediated cytotoxicity (Fig. 1D). Notably, DNTs effectively killed OCI-AML-3 and three primary AML samples (130783, 130794, and 090239) that were resistant to NK92 (Fig. 1D). Similarly, DNTs were more effective at lysing leukemic targets than ex vivo expanded primary CD8+ T cells (Supplementary Fig. S4A) and primary NK cells (Supplementary Fig. S4B).
The ability to generate large numbers of human DNTs allowed us, for the first time, to study their antileukemic activity in vivo. Using a patient-derived xenograft (PDX) model (Fig. 1E), we found that a single allogeneic DNT infusion significantly reduced the frequency of human CD45+CD33+ leukemic cells in the bone marrow of NOD scid gamma (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, NSG) mice inoculated with primary AML samples obtained from 4 different patients (Fig. 1F and G). Two additional DNT treatments further reduced leukemia load (Fig. 1H). Overall, 69 mice inoculated with primary AML blasts from 8 different patients, of which 42 received one dose of DNTs and 27 received three doses, had a 52.43% ± 30.31% and 74.35% ± 23.94% reduction in leukemia load, respectively, compared with the PBS-treated group. As inoculation of the patient AML samples that we have tested so far did not cause death in recipient mice even when engraftment reaches >80%, we were not able to study the effect of DNTs on recipient survival. Collectively, these results demonstrate that allogeneic DNTs can effectively target a broad range of primary AML cells both in vitro and in vivo in a dose-dependent manner.
Allogeneic DNTs can effectively target chemotherapy-resistant AML in vitro and in vivo
As chemotherapy resistance is the major cause of low survival rates in AML patients, we studied the effect of DNTs on chemotherapy-resistant AML cells. We found that 69.2% (9/13) of chemotherapy-resistant AML cells, obtained from chemotherapy-refractory or relapsing patients, were effectively killed by DNTs in vitro (Fig. 2A) and that their level of sensitivity to DNT-mediated cytotoxicity was comparable with those obtained from chemotherapy-susceptible patients (Fig. 2B). Consistent with this, significant reductions in the leukemia load were observed in mice inoculated with chemotherapy-resistant primary AML cells from 4 different patients after DNT cell treatment (Fig. 2C). Together, these results indicate that allogeneic DNTs are effective at targeting the majority of chemotherapy-resistant AML cells in vitro and in PDX models, supporting the use of these cells to eliminate AML cells that are not cleared by standard chemotherapy.
Infusion of DNTs does not cause GVHD or kill normal allogeneic PBMCs and CD34+ HSPCs
Allogeneic HSCT induces curative graft-versus-leukemia effects (10), but is associated with morbidity and mortality due to donor-derived immune cells attacking normal host cells and tissue (8, 9). To determine the potential toxicity of allogeneic DNTs toward normal cells, the susceptibility of normal PBMCs and CD34+ HSPCs to allogeneic DNT-mediated cytotoxicity was compared with that of primary AML patient samples and AML cell lines with similar maturation status (CD33+CD34− for PBMCs and CD33−CD34+ for HSPCs). DNTs displayed potent cytotoxicity against primary AML samples and AML cell lines but had virtually no cytotoxicity toward normal allogeneic PBMCs (Fig. 3A), peripheral blood CD33+ myeloid cells (Supplementary Fig. S5), or HSPCs (Fig. 3B).
To study whether DNTs have toxicities against normal tissues in vivo, DNTs or bulk human PBMCs were intravenously infused into NSG mice and monitored for associated morbidities. As expected from prior literature, infusion of human PBMCs caused severe xenogeneic acute GVHD (28) as evidenced by weight loss (Fig. 3C). Histologic analysis of PBMC-transfused mice revealed acute GVHD pathology in multiple organs, including portal inflammation in the liver, mononuclear infiltrates extending into the alveolar septa, endotheliitis in the lungs, and lamina propria expansion with some architectural distortion in the small intestine (Fig. 3D). However, despite tissue infiltration by DNTs, neither weight loss (Fig. 3C) nor tissue damage was observed in the liver, lungs, and intestines (Fig. 3D) even when equal or 4-fold higher numbers of DNTs compared with PBMCs were infused.
To further assess potential detrimental effects of allogeneic DNTs on normal HSPC engraftment and differentiation, NSG mice were engrafted with normal CD34+ HSPCs and subsequently treated with DNTs from two HDs that were allogeneic to the HSPC donors. Similar to reports by others (29, 30), we consistently observed a high level of chimerism from the HSPC donors within the spleen and bone marrow (∼70%–80%), and approximately 15% in the peripheral blood of engrafted mice. Also, no differences in the frequency or differentiation of hematopoietic cells derived from transplanted HSPC cells between DNT-treated and PBS-treated mice were observed (Fig. 3E and F), including the CD34+ HSPC population in bone marrow. These findings show that DNTs do not target allogeneic HSPCs and their progeny, nor interfere with differentiation of HSPCs into hematopoietic lineages. Together, these results demonstrate that ex vivo expanded allogeneic DNTs have potent antileukemic effects but are noncytotoxic to normal tissues and hematopoietic cells in xenograft models.
NKG2D and DNAM-1 contribute to DNT-mediated donor-unrestricted and leukemia-specific cytotoxic activity
Along with no associated toxicity, being able to use allogeneic cellular therapy without donor restriction will significantly increase its clinical applications. We found that DNTs from a single donor targeted blasts from different AML patients (Fig. 4A) and DNTs obtained from different donors showed comparable cytotoxicity with the same AML target (Fig. 4B), suggesting that the efficacy of DNT therapy does not depend on the donor origin and that the susceptibility of AML cells to DNTs is mainly determined by intrinsic characteristics of leukemic cells.
To dissect the mechanisms by which DNTs selectively recognize AML cells over normal cells in a DNT donor–unrestricted manner, we first studied the involvement of TCR. We found that the addition of αβ- and γδ-TCR blocking antibodies significantly reduced the level of cytotoxicity toward Jurkat cells, a T-cell lymphoma cell line, but did not affect DNT-mediated cytotoxicity against AML cells (Fig. 4C), supporting a TCR-independent recognition of AML cells by DNTs.
Next, we focused on innate receptor–ligand molecules involved in anticancer immunity. High expression of activating receptors NKG2D and DNAM-1 was observed on DNTs (Fig. 4D), and significantly higher expression of NKG2D ligands (ULBP1 and ULBP3) and DNAM-1 ligands (CD112 and CD155) was found on primary AML cells over normal PBMCs (Fig. 4E). Blocking NKG2D, DNAM-1, or both significantly reduced the ability of DNTs to kill primary AML cells and AML3/OCI cell line (Fig. 4F). In contrast, blocking other activating receptors, NKp30, NKp44, and NKp46, used by other immune cells to recognize cancer cells (31, 32), showed no effect on DNT–AML interactions (Supplementary Fig. S6). Collectively, these results demonstrate that DNTs can preferentially recognize and kill AML cells but not normal cells in a donor-independent manner, partially, through NKG2D and DNAM-1.
DNTs produce IFNγ upon encountering AML cells, which augments their cytotoxicity toward AML cells but not to normal PBMCs
Ex vivo expanded DNTs express a high level of intracellular IFNγ (Fig. 5A), but minimal IFNγ levels were detected in the supernatant from cocultures of allogeneic DNTs with normal PBMCs (0.50 ± 0.054 ng/mL) and DNT-resistant primary AML cells (0.28 ± 0.10 ng/mL). Interestingly, significantly higher levels of IFNγ (3.29 ± 0.58 ng/mL; Fig. 5B) were released when DNTs were cocultured with DNT-susceptible AML cells, which corresponded with the degree of cytotoxicity (Supplementary Fig. S7). The level of IFNγ release was significantly reduced in the presence of NKG2D and DNAM-1–blocking antibodies, further supporting the involvement of these molecules in the recognition of AML cells by DNTs (Supplementary Fig. S8). The addition of an IFNγ-neutralizing antibody significantly reduced AML cell death induced by DNTs (Fig. 5C), whereas addition of exogenous recombinant IFNγ (rIFNγ) resulted in a higher level of DNT-mediated cytotoxicity without direct toxicity toward AML cells (Fig. 5D-i and 5D-ii).
Pretreatment of DNTs with rIFNγ did not significantly affect their function (Fig. 5D-v), but incubation of AML cells with rIFNγ rendered them more susceptible to DNT-induced cytotoxicity (18.4% vs. 31.9% for untreated versus rIFNγ pretreated; Fig. 5D-iii and 5D-iv), demonstrating that IFNγ sensitizes the AML cells rather than augmenting the DNTs’ cytotoxic activity. Consistent with this, the level of DNT-mediated cytotoxicity significantly increased in 10 of 20 primary AML samples after rIFNγ pretreatment, including 4 of 6 otherwise DNT-resistant AML samples (Fig. 5E) and DNT-resistant AML cells were sensitized to a greater degree (Fig. 5F). Importantly, rIFNγ pretreatment did not affect the susceptibility of normal allogeneic PBMCs to DNTs (Fig. 5E and F).
IFNγ upregulates NKG2D and DNAM-1 ligand expression on AML cells
To understand the mechanism of how IFNγ sensitizes AML targets (Fig. 5D–F), we first tested the effect of IFNγ on NKG2D and DNAM-1 ligand expression given that these pathways contribute to DNT-mediated anti-AML activity. Although it has been reported that IFNγ downregulates expression of NKG2D ligands on solid tumours (33, 34), rIFNγ pretreatment upregulated the expression of NKG2D ligands ULBP1, ULBP2/5/6, ULBP3, and MICA/B, as well as DNAM-1 ligands CD112 and CD155, on AML cells (Fig. 6A). In contrast, IFNγ treatment did not affect the expression levels of NKG2D and DNAM-1 ligands on normal PBMCs (Supplementary Fig. S9), which is in agreement with IFNγ treatment lacking its effect on the susceptibility of PBMCs to DNTs (Fig. 5F and G). Furthermore, the effect of rIFNγ on AML susceptibility to DNTs, as shown in Fig. 5F, was neutralized by the blocking of NKG2D and DNAM-1 (Fig. 6B), confirming that IFNγ can exert its role through NKG2D and DNAM-1 pathways. Furthermore, the level of DNT-mediated cytotoxicity was inhibited by NKG2D and DNAM-1 antibodies at a significantly greater level for IFNγ-pretreated AML targets than untreated ones (22.69% ± 1.86% vs. 13.65% ± 0.68%; Fig. 6C). These data indicate that IFNγ increases the sensitivity of AML cells, but not normal PBMCs, to DNT-mediated cytotoxicity in part by upregulating NKG2D and DNAM-1 ligand expression on leukemic cells. It also revealed, for the first time, uniquely interconnected roles of NKG2D, DNAM-1, and IFNγ in AML cells, which form a positive feedback loop to facilitate DNT cell recognition and elimination of AML cells.
Previously, we showed that DNTs could be expanded from peripheral blood of a small number of AML patients during chemotherapy-induced complete remission and were able to kill autologous AML cells in vitro (26). Because of the low yield and purity of DNTs obtained from AML patients, it was not possible to study their function in vivo nor use them therapeutically. Herein, we developed a simple, cost-effective, highly reproducible method allowing for ex vivo expansion of DNTs from healthy individuals under GMP condition to therapeutic numbers with a high purity, in less than 20 days without feeder cells or genetic modifications. We demonstrated that the expanded DNTs selectively targeted a wide spectrum of primary AML samples both in vitro and in PDX models in a donor-unrestricted manner. Broad, yet cancer-specific cytotoxic activity of DNTs was also seen against cell lines derived from other forms of leukemia and lymphoma (Supplementary Fig. S10), such as Burkitt B-cell lymphoma (Daudi), T-cell lymphoma (Jurkat), histiocytic lymphoma (U937), chronic myeloid leukemia (K562), and myeloma (H929, 8226, and LDI), suggesting that our findings from this study may be translatable to other types of leukemia and lymphoma.
Importantly, although DNTs were cytotoxic to CD34+ AML cells in vitro and inhibited their engraftment in PDX models, they did not affect normal stem cell engraftment and differentiation. Contrary to the infusion of human CD4/CD8 T cells or PBMCs, infusion of human DNTs did not cause GVHD in recipients, demonstrating that DNTs selectively target leukemic cells while sparing normal cells and tissues. Moreover, we found that cryopreserved DNTs maintained comparable viability (Supplementary Fig. S11A) and antileukemia activity (Supplementary Fig. S11B) to those cultured without cryopreservation and thawing procedures. Given that ex vivo expanded allogeneic DNTs from HDs have no observed toxicity, can target broad range of leukemia cells in a donor-unrestricted manner and are cryopreservable, these cells can potentially be used as a new “off-the-shelf” cellular therapy for treating leukemia. Also, given their superior expansion profile, potent cytotoxic function, and lack of allo-response by endogenous TCRs, DNTs would be a good vehicle for CAR or transgenic TCR technology.
Mechanistically, we found that DNTs preferentially recognize and target AML cells in a TCR-independent and HLA-unrestricted manner that is partially mediated through the innate receptors, NKG2D and DNAM-1. Natural cytotoxic receptors, such as NKp30, NKp44, and NKp46, and other activating receptors, such as NKG2D and DNAM-1, are expressed by NK cells and subsets of activated T cells; a role for these molecules in cancer immunity has been shown previously (31, 35–38). The expression of at least one of NKG2D (39) and DNAM-1 (35) ligands has been detected in the majority of AML patients in large cohort studies. We found that blocking of NKG2D and DNAM-1, but not natural cytotoxic receptors, reduced the level of AML cell apoptosis induced by DNTs, indicating the role of these molecules in AML–DNT interaction. Although others have shown the role of NKG2D and DNAM-1 pathways in targeting leukemic cells, we are the first to show their unique positive feedback interaction with IFNγ. Notably, blocking these pathways did not completely abrogate the killing activity of DNTs; thus, there are likely other molecules involved in the selective recognition and targeting of AMLs by DNTs, which are being currently explored.
IFNγ is a well-known inflammatory cytokine with a pleiotropic function that can elicit both pro- and antitumorigenic effects (33, 40–43). A previous clinical trial using IFNγ as a monotherapy failed to achieve a clinical response in AML patients (44), which is in agreement of our finding that IFNγ alone does not induce AML cell death directly. However, we found that IFNγ renders AML blasts more susceptible to DNTs. Six primary AML blast samples obtained from 10 patients (60%) that were initially resistant to DNT-mediated cytotoxicity became susceptible after pretreatment with exogenous IFNγ. In combination with IFNγ, >85% (40/46) of tested AML samples were effectively killed by DNTs. Unlike solid tumors (33, 34), IFNγ induced higher expression of NKG2D and DNAM-1 ligands on AML cells, which rendered them more sensitive to DNTs and theoretically to other cytotoxic cells such as NK cells. Notably, normal PBMCs express low levels of NKG2D and DNAM-1 ligands, which were not upregulated by IFNγ, and rIFNγ treatment did not render normal cells sensitive to DNTs. These findings help to explain why DNTs can selectively target AML but not normal cells. Furthermore, our data suggest that rIFNγ and DNT combination therapy may result in a synergistic effect, leading to a greater efficacy against AML via sensitizing leukemic cells to DNT-mediated cytotoxicity and other modes of antileukemic activity of IFNγ (41–43, 45, 46).
Resistance to chemotherapy results in refractory AML and disease relapse, both of which significantly hamper clinical outcomes in patients. Alternative forms of treatment to target these cells are in urgent need. Here, we demonstrate that a majority of primary AML cells obtained from chemotherapy-resistant and relapsing patients are susceptible to DNT-mediated cytotoxicity both in vitro and in vivo. Expression of NKG2D and DNAM-1 ligands is regulated by the DNA damage repair pathway (47, 48), explaining higher levels of ligand expression on transformed cells (31, 37, 38, 49). The majority of chemotherapy drugs cause DNA damage and interrupt the cell cycle; hence, treatment of myeloma with doxorubicin and bortezomib has been shown to increase expression of NKG2D and DNAM-1 ligands (48, 50). These findings support the potential application of DNT therapy after conventional chemotherapy, which may yield synergistic effects via NKG2D and DNAM-1 pathways to target chemotherapy-resistant residual disease to prevent disease relapse.
We show in PDX models that 1 or 3 DNT infusions significantly reduced leukemia load, but complete eradication of AML cells may be needed to prevent relapse. A higher dose or more DNT treatments or DNT in combination with other forms of therapies may be needed to eradicate the disease. In this study, DNTs were infused 3 to 14 days after AML infusion when the leukemia load is relatively low. Whether DNTs given at later time points where recipients have a higher leukemia load are effective needs to be determined.
In summary, we demonstrate that allogeneic human DNTs have potent antileukemic activity against primary AML cells, including chemotherapy-resistant cells, both in vitro and in vivo in PDX models, without observed toxicity to normal cells and tissues and elucidate the underlying mechanism of the selective toxicity of DNTs toward AML. Therapeutically, our findings support the use of DNTs expanded from HDs as a new “off-the-shelf” nontoxic cellular immunotherapy to target chemotherapy-resistant AML populations following conventional chemotherapy to improve patient survival.
Disclosure of Potential Conflicts of Interest
J.E. Dick is a consultant/advisory board member for Trillium Therapeutics. L. Zhang is a consultant for WYZE Biotech. No potential conflicts of interest were disclosed by the other authors.
Conception and design: J. Lee, E. Streck, S.D. Der, J.E. Dick, L. Zhang
Development of methodology: J. Lee, E. Streck, L. Zhang
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Lee, M.D. Minden, W.C. Chen, B. Chen, A. Arruda, S. Kang, P. Achita, C. D'Souza, L. Zhang
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Lee, E. Streck, S. Kang, R.W. Childs, L. Zhang
Writing, review, and/or revision of the manuscript: J. Lee, M.D. Minden, B. Chen, D. Ly, C. D'Souza, R.W. Childs, J.E. Dick, L. Zhang
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Lee, M.D. Minden, B. Chen, H. Kang, Y. Li, R.W. Childs
Study supervision: J. Lee, J.E. Dick, L. Zhang
The authors thank Dr. Michael Cabanero for evaluation of GVHD histology, Dr. Thierry Mallevaey for providing CD1d-α-galactosylceramide, and all study participants. This work was supported by The Leukemia and Lymphoma Society Translational Research Program (grant # 6265-13), Canadian Cancer Society Research Institute (grant # 704121), and Canadian Institutes of Health Research Proof of Principle Grant Phase II (grant # 141723) to L. Zhang. L. Zhang is the Maria H. Bacardi Chair of Transplantation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.