Biomimetic Magnetosomes as Versatile Artificial Antigen-Presenting

Sep 18, 2017 - exhibited high performance for antigen-specific cytotoxic T-cell (CTL) expansion and stimulation but also visually ... KEYWORDS: biomim...
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Biomimetic Magnetosomes as Versatile Artificial Antigen-Presenting Cells to Potentiate T‑Cell-Based Anticancer Therapy Qianmei Zhang,†,‡ Wei Wei,§,‡ Peilin Wang,† Liping Zuo,† Feng Li,† Jin Xu,† Xiaobo Xi,§ Xiaoyong Gao,§ Guanghui Ma,§ and Hai-yan Xie*,† †

School of Life Science, Beijing Institute of Technology, Beijing 100081, P. R. China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China

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S Supporting Information *

ABSTRACT: Adoptive T-cell transfer for cancer therapy relies on both effective ex vivo T-cell expansion and in vivo targeting performance. One promising but challenging method for accomplishing this purpose is to construct multifunctional artificial antigen-presenting cells (aAPCs). We herein developed biomimetic magnetosomes as versatile aAPCs, wherein magnetic nanoclusters were coated with azide-engineered leucocyte membranes and then decorated with T-cell stimuli through copper-free click chemistry. These nano aAPCs not only exhibited high performance for antigen-specific cytotoxic T-cell (CTL) expansion and stimulation but also visually and effectively guided reinfused CTLs to tumor tissues through magnetic resonance imaging and magnetic control. The persisting T cells were able to delay tumor growth in a murine lymphoma model, while the systemic toxicity was not notable. These results together demonstrated the excellent potential of this “one-but-all” aAPC platform for T-cell-based anticancer immunotherapy. KEYWORDS: biomimetic, artificial antigen-presenting cells, targeting delivery, T-cell therapy expected in vivo.10 The study results on the survival of transferred CTLs in recipients vary widely; some groups even report no detectable CTLs immediately after transfer.11 As a result, few CTLs arrive at the cancer tissue, and the therapeutic effect is significantly compromised. To date, substantial efforts have been devoted to developing alternative strategies that can overcome the disadvantages and difficulties of using natural APCs.12 To improve the expanding efficiency of antitumor T cells, one important strategy is to construct artificial APCs (aAPCs), which are commonly realized by carefully modifying and decorating micro- or nanosized particles to tune their properties and signal presentation.13 For example, T. R. Fadel et al. constructed composite aAPCs by attaching T-cell stimuli onto bundled carbon nanotubes, which induced effective antigen-specific Tcell proliferation in vitro.14 Although this strategy allows for stringent control over the signals delivered, the conjugation on a solid support may lose some natural membrane functions (such as membrane fluidity and associated proteins), which are

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mmunotherapy has emerged as a promising therapeutic modality with the potential to significantly alter the cancer treatment paradigm.1,2 In cancer immunotherapy, the patient’s own immune system can be unleashed to fight cancer. Active immunotherapy is highly dependent on the efficient stimulation of antigen-specific immune cells, and adoptive Tcell transfer is one of the most important strategies.3−5 During this treatment, isolated autologous T cells are expanded and stimulated ex vivo by antigen-presenting cells (APCs), particularly dendritic cells (DCs), and then reinfused into the cancer patient to elicit potent antitumor responses.6,7 As the delivery of essential signals to selected subsets of antitumor T cells can be largely facilitated in vitro, this approach overcomes the limitations associated with vaccine-based strategies in cancer patients, who are often immune compromised.8 Although promising, T-cell-based strategies still suffer from several critical problems. First, the ex vivo expansion and stimulation of T cells with natural APCs is time-consuming and exhibits poor reproducibility.9 In a typical process, several months are required to produce therapeutic numbers of antigen-specific cytotoxic T cells (CTLs),5 and CTL quality control is difficult due to the variable DCs generated ex vivo. In addition, the survival and path of these cells are always less than © XXXX American Chemical Society

Received: July 13, 2017 Accepted: September 18, 2017 Published: September 18, 2017 A

DOI: 10.1021/acsnano.7b04955 ACS Nano XXXX, XXX, XXX−XXX

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ACS Nano also proved to have significant effects on T-cell activation.12 In this aspect, aAPCs that can closely mimic features of natural APCs may further improve their performance. Meanwhile, several strategies have been developed to improve the target affinities and circulation lifetimes of T cells. Typically, engineered T cells expressing either transgenic T-cell receptors or chimeric antigen receptors were constructed and showed promising results in the clinical.15 Similar to aforementioned aAPC approach, this strategy focuses on only part of the T-celltransfer therapy process.16 Therefore, constructing a multifunctional platform that can potentiate both ex vivo T-cell stimulation and expansion and in vivo tumor-targeting performance of T-cell-based anticancer therapy remains highly necessary but challenging. To this end, we herein constructed a biomimetic and versatile aAPC platform. In brief, magnetic nanoclusters (MNCs) with satisfactory superparamagnetism and magnetic response were coated with leukocyte membrane fragments (LMNCs). As the leukocyte membrane was pre-engineered with azide (N3) via intrinsic biosynthesis and metabolic incorporation of phospholipids, dibenzocyclooctyne (DBCO)modified T-cell stimuli could be decorated through mild and highly efficient copper-free click chemistry.17 In this way, peptide (SIINFEKL)-loaded major histocompatibility complex class-I (pMHC-I) and co-stimulatory ligand anti-CD28 (αCD28) for EG-7 tumors could be conjugated to the azidemodified LMNCs (N3-LMNCs) at a controllable density with good fluidity. The resultant biomimetic aAPC could not only efficiently expand and stimulate OT-1 CD8+ T cells ex vivo but also visually guided the reinfused CTLs to tumor tissues effectively through magnetic resonance imaging (MRI) and magnetic control. As a result, the tumor growth in the murineEG7-based model could be efficiently delayed with minor side effects, demonstrating the great promise of this “one-but-all” aAPC platform for T-cell-based anticancer immunotherapy (Scheme 1).

RESULTS AND DISCUSSION Construction of Magnetosome-Based aAPCs. To endow aAPCs with the aforementioned functions for MRI and magnetic control, the nanoparticles should be both superparamagnetic and magnetic. At a first glance, exhibiting these two properties seems impossible since they are contradictory in traditional nanoparticles.18,19 The nanoparticles (≈10 nm) commonly used to ensure superparamagnetism exhibit relatively low magnetization. Simply increasing the particle size can increase the saturation magnetization but also induces the superparamagnetic−ferromagnetic transition.20 To resolve this contradiction, we developed a hydrothermal approach to construct MNCs, which consisted of many ≈10 nm particulate building units. In such an architecture, the magnetic moment of individual clusters dramatically increased with the increasing particle size, while the superparamagnetism was maintained. The positive charges of branched polyethylenimine (PEI, Mw = 10,000) surfactant used in the MNC preparation facilitated the subsequent coating with negative charged membrane fragments, which could be verified by the formation of a uniform layer containing membrane protein components, the increased particle size, and the reversed zeta potential (Figure 1a−c). The saturation capacity of leukocyte membrane fragments for 100 μg MNCs was determined to be about 70 μg (Figure S1). Such a membrane coating significantly improved the particle stability in media and the biocompatibility with T cells (Figure S2). The saturation magnetization value of the MNCs was well maintained (more than 70 emu g−1), which enabled them to be collected by a commercial magnetic scaffold within 1 min (Figure 1e). Meanwhile, the MRI signal intensity was directly proportional to the particle concentration (Figure 1f), which suggested their potential for T2 contrast imaging. Having obtained the desired magnetosomes, we next decorated T-cell stimulatory signals through a copper-free click chemistry reaction. First, we modified the stimulatory signals (pMHC-I and anti-CD28) with DBCO, which could be demonstrated by the increased molecular weight (MALDITOF data in Figure S5). In a manner akin to the pristine stimulatory signals, such a modification showed little effect on their T-cell recognition capability21 (Figure 1g). Meanwhile, they could be effectively anchored to the leukocytes (Figure 1h) that were pre-engineered with azide by incubating the leukocytes with azide-choline (Figure S4). In this case, we accomplished N3-LMNC decoration via a copper-free click chemistry reaction, which could be confirmed by the colocalization of these two signals with MNCs (Figure 1i). The binding capacity could be tuned up to 50 μg N3-LMNCs to ∼3.4 μg DBCO-stimuli (Figure S5). Evaluations of Expansion and Stimulation of CTLs. Ex vivo expansion and stimulation are fundamental for T-cell-based therapy. With this in mind, we first evaluated these aspects of the performance of our biomimetic aAPCs. As shown in Figure 2a, the fold expansion of CD8+ T cells was gradually promoted with increasing aAPC doses. With a 100 μg dose (Fe concentration), for example, the fold expansion could reach up to 78-fold after 3 days of incubation, which was more than four times that obtained with soluble antibodies (soluble Ab) (Figure 2c) (**p < 0.01). Correspondingly, large cellular aggregates and significantly decreased intracellular CSFE levels (Figure 2b and Figure S6) could be observed, indicating that CD8+ T cells had adequately proliferated. After live/dead

Scheme 1. Construction of Biomimetic aAPC and Its Application in T-Cell-Based Anticancer therapy

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Figure 1. Construction of aAPCs. (a) (I) TEM images of MNCs; (II) TEM image of LMNC. Scale bar: 50 nm. The space between red lines indicated the leukocyte membrane. (b) Protein visualization of leukocytes (cell), leukocyte membranes (M), and LMNCs on SDS-PAGE followed by Coomassie staining. (c) Hydrodynamic sizes and surface zeta potentials of MNCs and LMNCs. (d) Cell viability of OT-1 CD8+ T cells treated with LMNCs or MNCs with different concentrations. (e) Magnetic hysteresis loop and rapid response of LMNCs. The saturation magnetization σs of LMNCs was determined to be 70.39 emu g−1. (f) T2 relaxation rate (1/T2, s−1) versus Fe concentration (mM) at 7 T. Inset: T2-weighted MRI images of LMNCs in 0.1% molten agar gel at different Fe concentrations. (g) Fluorescence statistics and corresponding CLSM images of OT-1 CD8+T cells sequentially incubated with primary Ab (antiCD28/pMHC-I or DBCO-antiCD28/DBCOpMHC-I) and fluorescence-labeled secondary Ab. (h) CLSM images of J774A.1 macrophages sequentially treated with DBCO-antiCD28/ DBCO-pMHC-I and corresponding fluorescence-labeled secondary Ab. Scale bars: 50 μm. (i) Fluorescence images of aAPC reacted with fluorescence-labeled secondary Ab, demonstrating the successful conjugation of the two stimuli. Scale bars: 5 μm. The results in (c) and (d) were the mean values from three independent experiments (n = 3).

those in the soluble Ab group. The expression levels could be further improved in the aAPC group, indicating that the fluidity in our biomimetic magnetosome played an improtant role. Correspondingly, the CTLs after aAPC treatment achieved the most potent cytotoxicity to target EG7 cells (Figure 2f and Figure S10). To further verify the activity of these CTLs, they were administered to EG7-tumor-bearing mice via a single intratumoral injection. With the same dose (1 × 106), tumorinfiltrating lymphocytes (TILs) per tumor gradually increased in the sequence of soluble Ab, fixed APC, and aAPC (Figure 2 g), again demonstrating the special features of the membrane. As a result, tumor growth was significantly delayed within 14 days in the aAPC group (Figure 2 h), while the effect on soluble Ab was moderate. Visually Targeting Performance of CTLs. Encouraged by the above results, we next evaluated the in vivo fate of these transferred CTLs. During the sample preparation, we observed another interesting phenomenon wherein the separated aAPCs could return to the CTL surface within 0.5 h (Figure S11),

staining, most T cells presented a bright green signal, reflecting their viability (Figure S8). We also noticed that expansion of CD8+ T cells decreased after precross-linking the membrane layer with glutaraldehyde (fixed aAPC). A possible explanation for this interesting phenomenon could be the important role of the membrane fluidity between the aAPC-T cell interactions.22 Our aAPC was constructed by coating the supporting material with natural cell membranes. When this biomimetic aAPC encountered T cells, the lateral diffusion of the membranes promoted the formation of receptor preclustering for T-cell recognition.23−25 Such a spatial organization would be restricted once the membranes were fixed. As a result, the interaction between aAPCs and T cells would be compromised (Figure S9) (*p < 0.05), leading to decreased T-cell expansion. The superior magnetism of our aAPCs made purifying the expanded T cells convenient, which facilitated our subsequent evaluation of the T-cell activation after different treatments. The expression levels of both IFN-γ (Figure 2d) and granzymeB (Figure 2e) in the fixed aAPC group were much higher than C

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Figure 2. In vitro expansion and stimulation of CTLs by aAPCs and verification of the CTL activity. (a) Fold expansions of CD8+ T cells with different aAPC concentrations. (b) Optical microscope imaging, Live (green)/Dead (red) staining, and CFSE dilution of T cells on day 3 at an aAPC dose of 100 μg (Fe concentration). Unstimulated control in gray. (c) Fold expansions on day 7 of CD8+ T cells under different conditions. The total concentration of Abs on fixed aAPC or aAPC was the same as that of soluble Abs. (d) IFN-γ released from the same amount of CTLs. (e) Normalized expression of intracellular granzyme-B in the same amount as that of CTLs activated by aAPC versus controls. (f) Cytotoxicity of CTLs on EG-7 cells. (g) Tumor-infiltrating lymphocytes (TILs) in tumors after intratumoral injection. (h) Delayed tumor growth in C57BL/6 mice previously inoculated with EG-7 for 6 days. Tumor volumes were normalized to volumes measured at day 6 for each group. The results in (a, c−g) were the mean values from three independent experiments (n = 3). The results in (h) were the mean values from six mice per group (n = 6) (*p < 0.05;**p < 0.01).

fluorescent markers and tracked them in vivo in real time. Regardless of the recruitment speed, accumulation amount, or retention period, the M-aAPC-CTL group always outperformed the aAPC-CTL group (Figure 3e and Figure S13), let alone the naked CTLs. All of these results demonstrated the satisfactory in vivo fate of the M-aAPC-CTLs. Anticancer Efficacy and Safety Evaluation. Finally, the antitumor efficacy was comparatively evaluated. Compared with the PBS control group, tumor growth was moderately suppressed in the CTL-reinfused group (Figure 4a) (**p < 0.01). The suppressive effect could be further enhanced in the aAPC-CTL group due to the improved tumor accumulation and CTL activity. Once a magnetic field was added, the target delivery performance in the M-aAPC-CTLs led to the best tumor restriction and survival rate (Figure 4b). Correspondingly, the infiltrated TIL and apoptotic cells in tumor tissues exhibited the following order: PBS < CTLs < aAPC-CTLs < MaAPC-CTLs (Figure 4c,d). In addition to the therapeutic effect, we also estimated the safety of the aAPC-based adoptive therapy.29 Considering the potential risk of a cytokine storm during traditional immunotherapy, we first evaluated two typical indicators, TNF-α and IL-6. Although their values increased, they were still in a safe range (Figure 4f,g), which could be attributed to the good targeting performance of our aAPC-CTLs. The typical biochemical markers, including aspartate aminotransferase

which might be attributed to the small size of the aAPCs and their good affinity with CTLs.26 This binding was sufficiently stable to endure the complex internal circulation process, since most aAPCs remained on the CTLs after intravenous injection (Figure 3c). The resulting aAPC-CTL complexes were therefore endowed with two special features. First, they could be well controlled in the magnetic field (Figure 3a), which could enrich more CTLs at the tumor site. Second, the T2 contrast signal intensity sourced from aAPCs showed an ideal linear relationship with the cell numbers (Figure 3b), which paved the way for subsequent in vivo MRI. To confirm this expected behavior, we comparatively investigated the in vivo fates of different CTLs after intravenous injection into tumor-bearing mice. As shown in the MRI images (Figure 3d), aAPC-CTLs showed superior tumor infiltration (indicated by the darker area) to that of CTLs alone (*p < 0.05). This could be attributed to the stealth effect of the coated macrophage membrane on MNC surfaces (Figure S12), which might endow the CTLs with longer circulation26,27 and therefore more opportunities to infiltrate the tumors.28,29 Once a magnetic field was added (M-aAPC-CTLs), the signal-tonoise ratio (SNR) value continued to decrease, demonstrating that more aAPC-CTLs were magnetically guided to the tumor. Correspondingly, the intratumoral MNC signal and Fe content were highest in the M-aAPC-CTL group. To gain deeper insights into these results, we also labeled the CTLs with D

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Figure 3. Tumor-targeting capability of CTLs in vivo. (a) In vitro magnetic field control of aAPC-CTL. Scale bars: 100 μm. (b) T2 relaxation rate (1/T2, s−1) and T2-weighted MRI imaging of aAPC-CTL complexes at different cell concentrations. (c) Optical microscopic images of aAPC-CTL complexes before and after transfusion and corresponding statistics of iron concentrations attached to CTLs. Red arrows indicate the preserved aAPC on CTLs after reinfusion. (d) Upper: representative in vivo T2-weighted MR images of EG-7-tumor-bearing mice after injection with different CTL-based formations and corresponding SNRs in tumor tissues. Red arrows indicate the accumulation of aAPCCTLs at the tumor site. Lower: representative CLSM images of tumor tissue sections and relative iron contents in tumors. Scale bars: 50 μm. The nuclei were counterstained with DAPI. The red color representing MNCs recorded at 630−635 nm. (e) Left: Visualization of the tumortargeting ability of different CTL-based formations at 6 h after intravenous injection with DIR-labeled CTLs. Images were representative of three independent experiments. Right: Statistics of total fluorescence intensity in tumor region at 6, 12, and 24 h. All bars represented means ± SD (n = 3). previously.18,30 The mouse monocyte macrophage cell line (J774A.1) was purchased from Beijing Xiehe Hospital. Anti-CD28 (LEAF Purified anti-CD28) and Alexa Flour 488-Conjugated Syrian Hamster IgG (H +L) were obtained from Biolegend. MHC-I (Dimer X1:Recombinant Soluble Dimeric Mouse H-2K[b]:Ig Fusion) and carboxyfluorescein succinimidyl ester (CFSE) were obtained from BD. Dulbecco’s modified eagle’s medium (DMEM), advanced RPMI 1640 medium, and fetal bovine serum (FBS) were obtained from Gibco. Rhodaminephalloidin and Hoechst 33342 were purchased from Life Technologies. Preparation and Characterization of LMNCs. The azidemodified leukocyte membrane fractions and the membrane-coated magnetic nanoclusters (LMNCs) were fabricated as previously reported.18 Briefly, J774A.1 cells were cultured with 0.1 mM azideCho in complete medium for 24 h to obtain azide-modified cells. Then the membrane fractions were purified through a discontinuous sucrose density gradient. After ultracentrifugation, the azide-modified leukocyte membrane fractions and magnetic Fe3O4 nanoclusters with diameter about 100 nm were mixed in Hepes C buffer at 4 °C under 20 r/min overnight. The size and surface zeta potential of LMNCs were measured by dynamic light scattering (Malvern ZEN 3600 Zetasizer). To study the stability, LMNCs were suspended in fetal bovine serum (Hyclone) or PBS (pH 7.4) at a final concentration of 100 μg/mL, and the zeta potentials were measured every day during 1 week. The membrane layer on the MNCs surface was visualized through transmission electron microscope (Zeiss Libra 120 PLUS EFTEM). Samples were deposited on a 400-mesh copper grid and negatively stained with vanadium before imaging. For SDS-PAGE protein analysis, the leukocyte cells, membrane fractions, and LMNCs were individually prepared in SDS sample buffer at an equivalent protein dose as measured by BCA kit. Then each sample was loaded into one well of a 10% SDS-polyacrylamide gel and ran at 120 V for 2 h. The relaxivities and R2 (spin−spin relaxation rate, 1/T2) were measured at 7 T using a 7T Bruker pharmascan animal instrument (Bruker Optics, Tsukuba, Japan). MR images of each phantom were

(AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), lactic dehydrogenase (LDH), and alkaline phosphatase (ALP), all fell in normal ranges after treatment (Table S1). No obvious temperature or weight change was observed (Figure 4e and Figure S14), and the hematoxylin and eosin (H&E) staining results from tissue sections did not show obvious inflammatory infiltrates or toxicity (Figure 4h). All of these data together convincingly demonstrated the in vivo biosafety of our biomimetic aAPCs for adoptive T-cell anticancer therapy.

CONCLUSIONS In conclusion, we developed a high-performance aAPC that could not only expand CTLs in vitro but also visually guide the reinfused CTLs to tumor tissues effectively in vivo. The aAPC was constructed by electrostatically coating MNCs with leucocyte membranes and then decorating the membranes with stimulatory signals through a copper-free click chemistry reaction. The spontaneous and mild construction strategy combined with the special characteristic of the membranes conferred the biomimetic aAPC with high activity and efficiency for CTL expansion and stimulation. By taking advantage of the satisfactory magnetization and T2 MR relaxation properties, the reinfused aAPC-CTLs could be delivered in a targeted fashion by magnetic control, and their accumulation could be monitored through MRI. Correspondingly, tumor growth was efficiently delayed with few side effects. These results together demonstrated that this multifunctional aAPC holds great promise for adoptive T-cell-based anticancer immunotherapy. MATERIALS AND METHODS Reagents and Materials. The Fe3O4 nanoclusters and azidecholine (azide-Cho) were prepared according to the methods reported E

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Figure 4. Therapy effect and safety evaluations. (a,b) Tumor volume change curves and survival percentages of mice. (c) Absolute numbers of TILs presented in tumors. (d) Apoptotic cells (green) detection by TUNEL immunofluorescence staining. Scale bars: 200 μm. (e) Temperature change curves of mice. Scale bar: 100 μm. (f, g) Serum TNF-α and α IL-6 statistics of different groups. (h) H&E stained tissue sections after treatment with M-aAPC-CTLs. Scale bar: 200 μm. All bars represented means ± SD (n = 6). obtained on a 7 T superconducting magnet with T2-weighted (3000/ 13.2) spin−echo sequences. Biocompatibility and Immunocompatibility Study of LMNCs. For biocompatibility study, the toxicity of MNCs or LMNCs on CD8+T cells was compared. Briefly, LMNCs or LMNCs were dispersed into advanced RPMI medium at a series of concentrations and then individually cocultured with CD8+T cells for 24 h. Cell viability was analyzed by exclusion of Trypan Blue dye. To study the immunocompatibility of LMNCs, 200,000 HUVEC cells or J774A.1 cells were treated with LMNCs or MNCs at a final concentration of 100 μg/mL for 24 h. Then, Alexa Flour 488conjugated phalloidin (L7528, Invitrogen) and Hoechst 33342 were added. Thirty min after, the cells were washed twice with PBS to remove excess particles prior to imaging. The iron content inside cells was analyzed with ICP. DBCO Modification of the Stimuli. A 50-fold molar excess of NHS-PEG4-DBCO (A134-10, ClickChemistryTools, USA) linker was added to anti-CD28 or MHC-I antibody and incubated at 4 °C overnight. Then the solution was filtered using a centrifugal filter device (Amicon Ultra-0.5, Millipore Co, German) at 7000 rpm at 4 °C for 30 min and suspended in PBS. The purified DBCO-Ab was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, AXIMA-Pertormance MA, SHIMADZU, Japan). Before application, the DBCO-MHC-I and SIINFEKL peptide were incubated at 1:64 molar ratio for 6 h at 37 °C,

and the reaction mixture was ultrafiltered in a 30 kDa ultrafiltration tube at 7000 rpm for 30 min at 4 °C. The retentate was peptide-loaded MHC-I (pMHC-I). Activity Verification of DBCO-Ab. Both the “click” reactivity and specific antigen recognization activity of the DBCO-modified stimuli were characterized. For “click” reactivity verification, azide-modified cells were plated and fixed with 4% paraformaldehyde at room temperature for 15 min. Then, DBCO-Ab or Ab at a final concentration of 20 μg/mL was added and incubated for 1 h at 37 °C. After washing, the fluorescent secondary antibody Alexa Flour 488-conjugated Syrian Hamster IgG (H+L) for anti-CD28, Alexa Flour 405-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) for pMHC-I was added. After incubation, the dishes were washed and observed under a confocal fluorescence microscope (Leica TCS SP5 laser scanning confocal microscope). Specific antigen recognization activity was characterized through fluorescence microscopy and flow cytometry. For fluorescence microscopy imaging, CD8+ T cells isolated from the spleen of OT-1 mice were plated on the 35 mm glass-bottom dishes. After incubation for 30 min, cells were fixed with 4% paraformaldehyde, blocked with 1% BSA. Then, DBCO-Ab or Ab at a final concentration of 5 μg/mL was added and incubated for 1 h at 37 °C. After which the fluorescent secondary antibody was added and incubated. For flow cytometry analysis, fresh purified CD8+ T cells of OT-1 mice at a density of approximate 106 cells were incubated with DBCO-anti-CD28/pMHC-I at a final concentration of 2 μg/mL for 1 F

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ACS Nano h at 37 °C. Then, the cells were stained by corresponding fluorescent secondary antibody. After washing, they were collected and stained with PE-labeled anti-CD8+ T-cell antibody, followed with wash, and then analyzed using a flow cytometer (CyAn ADP, Beckman Coulter, USA). Construction of aAPC. Equimolar amounts (0.03 nmol) of DBCO-pMHC-I and anti-CD28 were mixed in PBS and added to the LMNCs suspension (Fe: 50 μg) to react for 1 h at 37 °C under 30 r/ min. The resultant aAPC was washed with PBS to remove surplus stimuli. To prove the successful construction, LMNCs and aAPC were individually incubated with both two fluorescent secondary antibodies for 1 h at 37 °C under 30 r/min. After which they were washed three times with PBS by magnetic separation, and then observed under a fluorescence microscope. The fixed aAPC was prepared by incubating LMNCs with 0.1% glutaraldehyde at room temperature for 10 min before the DBCO-stimuli decoration. In Vitro Expansion and Stimulation of CTLs. Splenocytes were isolated from the spleen of OT-1 mice (aged 6−8 weeks) after depletion of erythrocytes by hypotonic lysis. CD8+T cells were isolated using a CD8 no-touch isolation kit, labeled with CFSE if necessary, and then extensively washed. The CD8+T cells were incubated in cell media composed of RPMI 1640 supplemented with FBS (10%), L-glutamine (1%), HEPES buffer (1%), nonessential amino acids, β-ME (0.1%), and penicillin (2%). For expansion and stimulation, 1 × 106 CD8+T cells and 100 μg of aAPC were mixed and incubated for 4−7 days in complete RPMI media. For control groups, equimolar amounts of soluble MHC-I and anti-CD28 (Soluble Ab) or aAPC cross-linked by glutaraldehyde (Fixed aAPC) were added into the culture system instead of aAPC. After 7 days of incubation, the mixture of cells and aAPC was collected by centrifugation, and then they were resuspend in PBS and repeatedly blowed by pipet to separate the colonies of cells from aAPC. Finally, a commercial magnetic column was used to remove the aAPC, with pure CTLs obtained. CFSE fluorescence was measured on day 4. Trypan Blue dye assay and a Live/Dead Viability/Cytotoxicity Kit were used to evaluate the viability of CD8+ T cells. To define the fold expansion of CTL proliferation, the CD8+T cells were cultured in the 96-well plate at the number of 5 × 105/mL (we defined the number as A). Then, a certain kind of stimulator (soluble Ab, fixed aAPC, or aAPC) was added to each well for T-cells expansion. During the whole period of expansion, the cell density was always kept at 5 × 105/mL by transferring the expansion solution to a new container with bigger and bigger capacity. In the end, all cells expanded from the original well were counted (we defined the number as B). The expansion fold was defined as the quotient of B divided by A. Lactate dehydrogenase (LDH) release assay (Roche, Penzberg, Germany) was used to determine the lytic activity of CD8+ T cells against EG-7 target cells in vitro according to the manufacturer’s guideline. Granzyme-B and IFN-γ Analysis. CD8+ T cells were collected, washed in Perm/Wash solution (BD Biosciences), and resuspended in 250 μL of Cytofix/Cytoperm (BD Biosciences) solution for 20 min at 4 °C. Then, the cells were washed and resuspended in the staining solution of antigranzyme-B PE-Cy7 (Biolegend) at a ratio of 1:400 for 30 min at 4 °C. After incubation, cells were washed and then resuspended in 4% PFA before measurement. Flow cytometry measurements were performed using a Multisizer 3 (Beckman Coulter) with appropriate compensation and staining controls. Fluorescence analysis was performed using FlowJo software (Tree Star, Ashland, OR) and gating on CD8+ subsets in side scatter (SSC) vs forward scatter (FSC) plots. Granzyme-B mean fluorescence was normalized to the mean fluorescence measured in aAPC-activated T cells. For IFN-γ analysis, the supernatant of the cells was collected and analyzed by ELISA assay according to the manufacturer’s guidelines. Formation of aAPC-CTLs Complex and MRI Imaging Capability Confirmation. The aAPC-CTLs complex was formed by incubating CTLs with aAPC at a concentration of 20 pg Fe/cell for 0.5 at 37 °C. Then they were collected and divided into six groups with different numbers (0.124 × 105, 0.256 × 105, 0.64 × 105, 1.6 × 105, 4 × 105, and 10 × 105). Complex of each group was diluted in 0.1% molten agar gel. Agar gel without cells was recruited as control.

The T2-weighted images of aAPC-CTLs complex were acquired with a conventional spin−echo (SE) acquisition with TE values ranging from 6.3 to 500 ms (TR = 3000 ms). Signal intensity at different TE was measured to calculate the T2 value of each tube (7T, spin−echo acquisition, TR = 3000 ms, TE = 50 ms). In Vivo Tumor Targeting Evaluation of aAPC-CTLs. CTLs were labeled with DIR, and some of the labeled CTLs were further incubated with aAPC to form aAPC-CTLs complex. A subcutaneous transplantable model of EG-7 was established by inoculating EG-7 cells (1 × 106 cells) in the right flank of each mouse. Then, all the EG7 tumor-bearing C57 mice were randomly assigned into three groups and individually treated with labeled CTLs or aAPC-CTLs at a number of 2 × 106 in 100 μL of PBS by intravenous injection. The third group was treated with both aAPC-CTLs and an additional magnetic field on the right flank (M-aAPC- CTLs). Tumor targeting capability of injected CTLs was visualized by in vivo MRI imaging and fluorescence imaging. MRI scan was performed at 12 h after reinfusion of CTLs. Echo signals of the tumors were acquired with T2-wighted fast spin−echo sequence (TSE, TR = 3000 ms, TE = 50 ms, matrix = 256 × 256, slice thickness = 8/18, field of vision = 35 × 35 mm, flip angle = 90°). The total fluorescence intensity in tumor area was calculated using an In Vivo Imaging System (FX Pro, Kodak, Japan). For resected organ imaging, the animals were euthanized, and the tumors and organs were excised and imaged with the same imaging system. In Vivo Anticancer Efficacy Evaluation. C57BL/6 mice were inoculated subcutaneously with 4 × 105 EG-7 cells on the right flank prior to treatment. When tumors were palpable, mice were randomly subdivided into four groups: (1) no treatment (control), (2) magnetically targeted aAPC-CTLs (M-aAPC-CTLs), (3) aAPCCTLs, and (4) CTLs. Tumor growth of each group was monitored at 2 day intervals using digital calipers, with volume calculated using an ellipsoid approximate, volume = 1/2 length × width2. The body weight and temperature were monitored every day. To evaluate the apoptosis levels in tumor cells, tumor tissue sections were stained by terminal deoxynucleotidyl transferased dUTP nick end labeling (TUNEL) assay (FragFL DNA Fragmentation Detection kit, Merck, Germany) according to the manufacturer’s protocol. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA) and observed by confocal microscopy imaging. For quantitative analysis of apoptotic levels, a total of 100 nuclei over two separate slides were taken into account. TILs Isolation. Subcutaneous EG-7 tumors were resected from mice, weighed, minced, and then placed in serum-free RPMI media containing 175 U/mL of Collagenase IA (Sigma). The tissue suspension was incubated at 37 °C for 1 h and passed through a 200 mesh tissue filter. After washing, the pellet was resuspended in 0.5 mL of media and overlaid over mouse lympholyte-M media (Solarbio) for lymphocyte isolation according to the manufacturer’s guideline, followed by centrifugation at 1500 rpm. The resulting buffy coat layer was removed, washed, and subsequently resuspended in 1 mL of staining buffer. All cell suspensions were counted to determine the absolute numbers of isolated TILs. Safety Estimation of the aAPC-Based Adoptive Therapy. For humane reasons, animals were sacrificed when the implanted tumor volume reached 1500 mm3. The tissue sections of the hearts, livers, spleen, lung, and kidneys were stained with H&E (hematoxylin/eosin) and analyzed by light microscopy for post-mortem histopathology analysis. The serum levels of urea nitrogen (BUN), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were analyzed spectrophotometrically using an automated analyzer (Hitachi-917, Hitachi Ltd., Tokyo, Japan). The serum cytokines were analyzed on day 18 using commercially available enzyme-linked immunosorbent assay kits. All animal experiments were performed in compliance with the guide of care and use of laboratory animals. Statistical Analysis. Statistical evaluations of data were performed using the Student’s t test. All of the results were expressed as mean ± standard error unless otherwise noted; *P < 0.05, **P < 0.01. G

DOI: 10.1021/acsnano.7b04955 ACS Nano XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b04955. Additional figures and tables (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Hai-yan Xie: 0000-0002-6330-7929 Author Contributions ‡

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (nos. 81571813, 21372028, 21422502, 81302704, and 21622608), National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (2014ZX09102045-004), National Key R&D Program of China (2017YFA0207901), and Beijing Talents Fund (2015000021223ZK20). REFERENCES (1) Mellman, I.; Coukos, G.; Dranoff, G. Cancer Immunotherapy Comes of Age. Nature 2011, 480, 480−489. (2) Ye, Y. Q.; Wang, J. Q.; Hu, Q. Y.; Hochu, G. M.; Xin, H. L.; Wang, C.; Gu, Z. Synergistic Transcutaneous Immunotherapy Enhances Antitumor Immune Responses Through Delivery of Checkpoint Inhibitors. ACS Nano 2016, 10, 8956−8963. (3) Maus, M. V.; Fraietta, J. A.; Levine, B. L.; Kalos, M.; Zhao, Y.; June, C. H. Adoptive Immunotherapy for Cancer or Viruses. Annu. Rev. Immunol. 2014, 32, 189−225. (4) Fesnak, A. D.; June, C. H.; Levine, B. L. Engineered T Cells: the Promise and Challenges of Cancer Immunotherapy. Nat. Rev. Cancer 2016, 16, 566−581. (5) Coulie, P. G.; van den Eynde, B. J.; van der Bruggen, P.; Boon, T. Tumour Antigens Recognized by T Lymphocytes: at the Core of Cancer Immunotherapy. Nat. Rev. Cancer 2014, 14, 135−146. (6) Butler, M. O.; Hirano, N. Human Cell-Based Artificial AntigenPresenting Cells for Cancer Immunotherapy. Immunol. Rev. 2014, 257, 191−209. (7) Gattinoni, L.; Klebanoff, C. A.; Restifo, N. P. Paths to Stemness: Building the Ultimate Antitumour T Cell. Nat. Rev. Cancer 2012, 12, 671−684. (8) Shao, K.; Singha, S.; Clemente-Casares, X.; Tsai, S.; Yang, Y.; Santamaria, P. Nanoparticle-Based Immunotherapy for Cancer. ACS Nano 2015, 9, 16−30. (9) Marrache, S.; Tundup, S.; Harn, D. A.; Dhar, S. Ex Vivo Programming of Dendritic Cells by Mitochondria-Targeted Nanoparticles to Produce Interferon-Gamma for Cancer Immunotherapy. ACS Nano 2013, 7, 7392−7402. (10) Meidenbauer, N.; Marienhagen, J.; Laumer, M.; Vogl, S.; Heymann, J.; Andreesen, R.; Mackensen, A. Survival and Tumor Localization of Adoptively Transferred Melan-A-Specific T Cells in Melanoma Patients. J. Immunol. 2003, 170, 2161−2169. (11) Heslop, H. E.; Ng, C. Y.; Li, C.; Smith, C. A.; Loftin, S. K.; Krance, R. A.; Brenner, M. K.; Rooney, C. M. Long-Term Restoration of Immunity Against Epstein-Barr Virus Infection by Adoptive Transfer of Gene-Modified Virus-Specific T Lymphocytes. Nat. Med. 1996, 2, 551−555. H

DOI: 10.1021/acsnano.7b04955 ACS Nano XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsnano.7b04955 ACS Nano XXXX, XXX, XXX−XXX