Interleukin-2 Functionalized Nanocapsules for T ... - ACS Publications

Oct 10, 2016 - For this purpose, defined amounts of azide-functionalized IL-2 were linked ... and murine T cell populations with various IL-2 receptor...
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Interleukin‑2 Functionalized Nanocapsules for T Cell-Based Immunotherapy Stefanie U. Frick,†,§ Matthias P. Domogalla,†,‡,§ Grit Baier,‡,§ Frederik R. Wurm,‡ Volker Mailan̈ der,†,‡ Katharina Landfester,*,‡,§ and Kerstin Steinbrink*,†,§ †

Department of Dermatology, University Medical Center Mainz, Johannes Gutenberg-University Mainz, Mainz D-55099, Germany Max Planck Institute for Polymer Research, Mainz D-55128, Germany

‡

S Supporting Information *

ABSTRACT: A major demand on immunotherapy is the direct interference with specific immune cells in vivo. In contrast to antibodyengineered nanoparticles to control dendritic cells function, targeting of T cells for biomedical applications still remains an obstacle as they disclose reduced endocytic activities. Here, by coupling the cytokine interleukin-2 (IL-2) to the surface of hydroxyethyl starch nanocapsules, we demonstrated a direct and specifc T cell targeting in vitro and in vivo by IL-2 receptor-mediated internalization. For this purpose, defined amounts of azide-functionalized IL-2 were linked to alkyne-functionalized hydroxyethyl starch nanocapsules via copper-free click reactions. In combination with validated quantification of the surface-linked IL-2 with anthracen azide, this method allowed for engineering IL-2-functionalized nanocapsules for an efficient targeting of human and murine T cell populations with various IL-2 receptor affinities. This nanocapsulemediated technique is a promising strategy for T cell-based immunotherapies and may be translated to other cytokinerelated targeting systems. KEYWORDS: nanocapsules, T cells, interleukin-2, click chemistry, hydroxyethyl starch, immunotherapy, human, murine CD8+ T cells.14 Recent investigations ascertained that regulatory T cells were more prone to IL-2 interaction and low-dose administration of IL-2 was sufficient for regulatory T cell stimulation. In contrast, other T cell populations required higher doses for activation as used in FDA approved cancer immunotherapies.15−17 Thus, IL-2 functionalization of nanocarriers will allow targeting of specific T cell subpopulations depending on the amount of coupled IL-2.

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lthough dendritic cells are the key players in immune induction, current dendritic cell-based immunotherapies display limited success rates in cancer.1,2 This is particularly due to the immunosuppressive part of the immune system resulting in a nonefficient T cell response.3,4 In addition, T cells play pivotal roles in the pathogenesis of autoimmunity, inflammatory and allergic disorders, and transplant rejections.5−8 Immunosuppressive and modulatory drugs affecting T cell functions are very efficacious in the therapy of inflammatory diseases. However, many of these treatments are accompanied by pronounced side effects.9 Therefore, T celltargeting by nanoparticles is rising as an innovative strategy in immunotherapy for cancer and inflammatory immune reactions, and a T cell-based immunotherapy by nanocarriers may efficiently improve clinical therapeutic approaches.10,11 The cytokine interleukin-2 (IL-2) can interact with the high affinity IL-2 receptor (trimeric, composed of CD25 [IL-2Rα], CD122 [IL-2Rβ], CD132 [IL-2Rγ]) or the low affinity IL-2Rβγ on T cells. In an initial step, IL-2 binds to IL-2Rα. After recruitment of IL-2Rβ/IL-2Rγ, the entire IL-2-/IL-2R complex is internalized.12,13 CD25 is highly present on regulatory CD4+FoxP3+ T cells and inducible on stimulated CD8+ and CD4+ effector T lymphocytes, whereas the dimeric IL-2 ̈ and memory receptor is expressed on NK cells and naive © 2016 American Chemical Society

RESULTS AND DISCUSSION In our study, we linked human IL-2 (15 kDa four α-helical bundle cytokine), which interacts with the human as well as the murine IL-2R, to the surface of hydroxyethyl starch (HES) nanocapsules. In previous studies, unfunctionalized HES nanocapsules showed no specific uptake by cells.18 Biocompatible HES nanocapsules were synthesized using the inverse water-in-oil miniemulsion polymerization that allows the encapsulation of small molecules and biological macromolecules (Figure S1a). For detection of the nanocapsules by flow cytometry and confocal laser scanning microscopy, the Received: December 17, 2015 Accepted: October 10, 2016 Published: October 10, 2016 9216

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Figure 1. Synthesis and characterization of HES-D-IL-2 nanocapsules. (a) Azide-functionalized IL-2 was covalently linked to DBCOfunctionalized HES-D nanocapsules via copper-free click reaction. Physicochemical analysis and schematic structure of IL-2-functionalized HES-D-IL-2 (red) and the corresponding control capsules, including HES (green) and HES-D (blue). Capsule diameter (Dz) and zeta potential were analyzed using DLS and electrophoretic mobility (Zeta Nanosizer), respectively (relative standard deviation (rSD) and standard deviation (SD) are depicted next to the measurements). Relative fluorescence intensity (SR101 of capsules) was analyzed via fluorescence spectrometry; n = 3. (b) SEM (upper panel) and TEM (lower panel) images of HES-D-IL-2 nanocapsules. Scale bar represents 200 nm. (c) HES-D and HES-D-IL-2 titration on IL-2-dependent murine CTLL-2 cells. Proliferation was assessed by [3H]-thymidine incorporation, and counts per minute (cpm) were plotted against concentration. One representative experiment out of 4 with mean ± SD from triplicates is depicted.

less than half of the fluorescence signal in comparison with control capsules (Figure 1a). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images demonstrated spherical nanocapsules with a homogeneous size distribution (Figure 1b). Biological activity of the nanocapsulebound IL-2 was ascertained by using IL-2-dependent murine CTLL-2 cells, demonstrating a dose-dependent induction of T cell proliferation (Figure 1c). Relevant amounts of soluble IL-2 in the supernatants of the nanocapsules were excluded in comparison to the effect of HES-D-IL-2 induced T cell proliferation (Figure S2b). The biological activity of the nanocapsule-bound IL-2 for inducing T cell proliferation was assessed by a direct comparison of calculated IL-2 molecules present on the capsule surface with the same amount of soluble, unmodified IL-2 molecules. We observed a similar T cell activation (CTLL-2) in both experimental settings, excluding a relevant loss of biological activity of the nanocapsule-coupled cytokine (Figure S2c). HES-D-IL-2 induced murine CTLL-2 proliferation already indicated capsule internalization, as IL-2 incorporation was found to be required for T cell proliferation, whereas the sole binding of IL-2 to IL-2R does not induce proliferation.20

HES nanocapsules were labeled with the fluorescent dye sulforhodamine 101 (SR101). They were surface functionalized with dibenzylcyclooctyne (DBCO)-PEG5-NHS ester binding to NH2 groups present on the carriers’ surface to introduce an active alkyne group and are referred to as “HES-D” (Figure S1b). The cytokine IL-2 was azide functionalized at its Nterminus using NHS-PEG5-N3 ester chemistry (Figure S1b−d), and the biological activity was approved by induction of T cell proliferation of IL-2-dependent murine CTLL-2 cells compared to soluble, unfunctionalized IL-2 (Figure S2a). Subsequently, IL-2 was linked to DBCO-functionalized HES capsules via copper-free click reaction for obtaining “HES-D-IL-2” nanocapsules (Figure S1b).19 The average size of redispersed HES nanocapsules was about 210 nm and coupling of DBCO groups and IL-2 did not change the diameters (220 and 215 nm, respectively, Figure 1a). Control HES nanocapsules revealed a zeta potential of −19 mV, whereas IL-2 conjugated HES nanocapsules showed a zeta potential of −7 mV, considerably closer to neutrality (Figure 1a). Comparing the relative fluorescence intensities of the encapsulated dye by normalizing to HES-D capsules with the highest SR101 signal, HES-D-IL-2 capsules were found to bear 9217

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Figure 2. Uptake and proliferative potential of HES-D-IL-2 nanocapsules. (a) Activated CD4+CD25+ T cells were incubated with HES-D and HES-D-IL-2 for 72 h, and percentages of nanocapsule positive cells were measured using flow cytometry. The nanocapsule incorporated dye SR101 (y-axis) was plotted against CellTrace Violet (x-axis). Dilution of CellTrace Violet depicts IL-2 induced proliferation of activated T cells. Representative data from 8 independent experiments are shown. (b) SR101+ nanocapsule uptake after 72 h incubation of 8 independent experiments is summarized in the graph illustrating mean ± SD. (c) Anti-CD3/CD28 mAb activated CD4+CD25+ T cells were incubated with HES-D-IL-2 at 75 μg·mL−1 for 72 h. Cell membrane was stained with CellMask Deep Red (in red), and nucleus was visualized using Hoechst dye (in blue) before conducting confocal laser scanning microscopy. SR101 signal (in green) was amplified in all images equally (contrast 50%) for better visualization, and intracellular SR101+ nanocapsules are marked with white arrows. Scale bar represents 5 μm. (d) IL-2 induced proliferation is visualized by the relative division index that was calculated as the ratio of the division index of nanocapsule treated CD4+CD25+ T cells to control cells treated with 50 U mL−1 IL-2 alone. Mean ± SD of 5 independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001.

presence or absence of IL-2) were found to be predominantly associated with the cell membrane, suggesting that the control nanocapsules preferentially bind to the surface of human T cells (Figure S3d). In addition, proliferation inductive potential of HES-D-IL-2 nanocapsules compared to control capsules is depicted by the division index (Figures 2d and S4). A toxic effect induced by control or IL-2-functionalized nanocapsules was excluded by analysis of dead cells (Figure S5). The IL-2 nanocapsules studied here exhibited a size of about 200 nm, allowing the internalization of the IL-2 nanocapsules/IL-2R complex as demonstrated by cLSM and functional T cell proliferation assays. We chose this size as previous analyses had disclosed that uptake of the IL-2/IL-2R complex is a clathrinindependent mechanism which is dominantly used by cargoes that are ≥200 nm in size.21,22 However, clustering and crosslinking of the IL-2 receptor induced by binding of IL-2functionalized nanocapsules can change the receptor-mediated endocytic mechanisms as well as the favorable size of particles endocytosed by T cells. In order to confirm the specificity of the uptake of IL-2functionalized nanocapsules, we performed blocking experiments using the antihuman CD25-mAb basiliximab.23 In the

In order to analyze the uptake and biological activity of HESD-IL-2 on primary human T cells, CD4+ T cells were isolated from peripheral blood, followed by anti-CD3 and anti-CD28 mAb activation for upregulation of CD25 expression and staining with the cell proliferation dye CellTrace Violet (Figure S3a). Flow cytometry analysis demonstrated that SR101+ HESD-IL-2 capsules showed a significantly increased uptake by human CD4+CD25+ T cells compared to HES-D control capsules (Figure 2a, b). CellTrace Violet costaining revealed a vigorous T cell proliferation induced by HES-D-IL-2 capsules (Figure 2a) pointing to an internalization of the HES-D-IL-2/ IL-2R complex. Moreover, incubation of T cells with control HES or HES-D capsules together with IL-2, resulting in activated and proliferating T cells, revealed significantly increased percentages of HES-D-IL-2 capsule positive T cells compared to both control capsules (Figure S3b,c). Thus, these data indicate that HES-D-IL-2 nanocapsules were presumably incorporated by an IL-2 receptor specific mechanism, as IL-2/ IL-2R complex internalization is critically required for induction of T cell proliferation.20 The intracellular uptake of HES-D-IL-2 capsules was confirmed by confocal laser scanning microscopy (Figures 2c and S3d), whereas control HES-D capsules (in the 9218

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Figure 3. CD25-dependent uptake of HES-D-IL-2 nanocapsules. Proliferation of CD4+CD25+ T cells incubated with HES-D-IL-2 nanocapsules supplemented with or without basiliximab was assessed by (a) [3H]-thymidine incorporation (one representative experiment from 5 independent experiments is shown as mean ± SD derived from triplicates) and by (b) flow cytometry analysis measuring the extent of CellTrace Violet dilution (x-axis). Pooled data (left panel) of 5 independent experiments with mean ± SD and representative histograms of one experiment (right panel) are shown. (c) Uptake of SR101+ HES-D-IL-2 nanocapsules by CD4+CD25+ T cells supplemented with or without 20 μg·mL−1 of the antihuman CD25 antibody basiliximab was assessed by flow cytometry analysis. Summarized data (left panel) of 5 independent experiments as percentages (upper left) and number (lower left) of SR101+ T cells (as means ± SD) and representative histograms of one experiment (right panel) are shown *p < 0.05, **p < 0.01.

(anth-N3).25 The fluorescence of anth-N3 is quenched (via an electron transfer) leading to an increased quantum yield (48fold).26 Numbers of the reacted azide groups on the surface can be assessed from the fluorescent enhancement allowing the calculation of the amounts of surface-coupled IL-2 (Figure 4a).25 Functional investigations revealed that incubation of murine CTLL-2 with HES-D-IL-2/2 (2-fold lower IL-2) and HES-D-IL-2/10 (10-fold lower IL-2) capsules resulted in a dosedependently reduced T cell proliferation as compared to regular HES-D-IL-2 (Figure S7b). In line with these experiments, HESD-IL-2/2 and HES-D-IL2/10 nanocapsules induced a significantly diminished proliferation of human CD4+CD25+ T cells (Figure 4b) due to a significantly impaired uptake of HES-DIL-2/2 and HES-D-IL2/10 nanocapsules (reduction in percentage and MFI) (Figure 4c). To test the uptake of HES-D-IL-2 nanocapsules by different ̈ T cell populations, we additionally investigated human naive,

presence of the antibody, the proliferation of human CD4+CD25+ T cells, induced by IL-2-functionalized nanocapsules, was significantly and dose-dependently inhibited resulting in a complete abrogation at lower HES-D-IL-2 doses (Figure 3a,b). In line with these results, uptake of HES-D-IL-2 capsules (but not of control HES-D, Figure S6) by human CD4+CD25+ T cells was blocked through basiliximab, as demonstrated by reduced percentage and absolute number of HES-D-IL-2 positive T cells (Figure 3c). As described above, binding and internalization of IL-2 by T cells depends on the dose of the cytokine and the expression and affinity of the specific IL-2R complex.16,17,24 Therefore, we generated HES nanocapsules with defined ratios of bound IL-2 by controlling the degree of functionalization as well as by assessing the amounts of surface-bound IL-2 (Figure S7a). Alkyne groups of HES-D capsules were quantified by the quantitative click reaction with 9-(azidomethyl)anthracene9219

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Figure 4. Functionalized HES nanocapsules with different amounts of surface linked IL-2. (a) NH2 groups per nanocapsule (mean ± SD) were assessed with titration experiments on a particle charge detector by titrating the amine groups against the negatively charged polyelectrolyte poly(ethylene sulfonate) (PES-Na) to define the point of zero charge (the same nanocapsules were used for all three samples). The amount of DBCO groups, IL-2 molecules, and IL-2 (mg × 10−16) present on the capsule surface was quantified using 9-(azidomethyl)anthracene (mean ± SD). (b) Proliferation of human CD4+CD25+ T cells induced by HES-D-IL-2, HES-D-IL-2/2, and HES-D-IL-2/10 was detected by (b) [3H]thymidine incorporation depicted as counts per minute (cpm) (left panel) and by flow cytometry analysis assessing CellTrace Violet positive T cells (right panel). One representative experiment (left panel) and pooled data of 3 independent experiments (right panel) are depicted. (c) Uptake of HES-D-IL-2 nanocapsules was detected by flow cytometry. Percentage of SR101+ T cells normalized to 50 U·mL−1 IL-2 induced T cell number (=100%) (left panel) and mean fluorescence intensity (MFI) of SR101+ T cells (right panel) are shown. Summarized data of 4 experiments are demonstrated. *p < 0.05, **p < 0.01, ***p < 0.001.

nonactivated CD4+CD25− (CD45RA+CD45RO−) T cells and CD4+CD25high (FoxP3+) regulatory T cells (exhibiting a high suppressive capacity as previously shown)27 in comparative studies with activated CD4+CD25+ T cells (phenotype analysis: Figures 5a and S8a). Comparing the uptake between activated ̈ human CD4+ T cells revealed a significantly elevated and naive internalization of HES-D-IL2 and HES-D-IL-2/2 (2-fold lower IL-2) nanocapsules by activated human CD4+CD25+ T cells (Figure 5b [percentage], Figure S8b [MFI]). Intriguingly, we found the highest percentage of HES-D-IL-2 positive T cells

within the population of CD4+CD25high regulatory T cells ̈ and activated T cells (Figures 5b and S8b compared to naive [MFI]). However, in contrast to activated CD25+ (Figure 4c, ̈ CD25− T cells (Figure 5c, left panel), the left panel) and naive uptake by regulatory CD25high T cells was not further reduced by decreasing amounts of HES-D-IL-2, HES-D-IL-2/2, and HES-D-IL-2/10 nanocapsules (Figure 5c, right panel). Additional kinetic studies of regulatory T cells after 4 and 24 h were performed which confirmed these results and excluded a timedependent effect (Figure S9a). These data indicate that even 9220

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̈ CD4+CD25− vs activated CD4+CD25+ or regulatory CD4+CD25high human T cells. (a) Figure 5. Uptake of HES-D-IL-2 nanocapsules by naive Isolated human T cell populations are depicted for their CD25 expression and plotted against CD4. (b) The in vitro uptake of IL-2̈ CD4+CD25−, activated CD4+CD25+, and functionalized nanocapsules (HES-D-IL-2, left panel) and (HES-D-IL-2/2, right panel) by naive + high regulatory CD4 CD25 human T cells was assessed by flow cytometry analysis after 72 h. (c) Uptake of 1, 25, and 75 μg HES-D-IL-2, HES̈ (left panel) and regulatory (right panel) human CD4+ T cells after 72 h. Capsule positive (SR101+) T D-IL-2/2, and HES-D-IL-2/10 by naive cells are depicted as mean percentage ± SD (pooled data of 4 independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant.

To determine the in vivo uptake of the nanocapsules in secondary lymphoid organs, we i.v. injected groups of wild-type C57BL/6 mice with control HES-D and HES-D-IL-2. Cellspecific costaining of lymph node-related immune cells revealed significantly increased percentages of HES-D-IL-2 positive CD4+CD25+ T cells and CD8+CD25+ T cells compared to unfunctionalized control capsules (Figure S10a). We did not find any significant differences analyzing the uptake by B cells (B220+) or antigen-presenting cells like CD11c+ dendritic cells, CD11b+ myeloid cells, or F4/80+ macrophages (Figure S10b), demonstrating a T cell specific targeting in vivo as well. As our study predominantly focuses on human T cells, we established an in vivo model which allows the analysis of the uptake of nanocapsules by human T cells in vivo. For this purpose, immunodeficient Rag2−/−γc−/− mice lacking murine T and B cells were reconstituted with human CD4+ T cells

the low IL-2 amounts of HES-D-IL-2/10 nanocapsules did not limit the CD25-dependent internalization of the nanocapsules due to the strong IL-2 receptor expression of regulatory T cells. A direct comparative analysis of the uptake of 25 μg of HES-D̈ or activated vs IL-2, HES-D-IL-2/2, and HES-D-IL-2/10 by naive regulatory T cells, respectively, depicted that the significantly elevated uptake by regulatory T cells became more pronounced at reduced IL-2 concentrations through which activated T cells ̈ T cells were hardly affected were significantly less and naive (Figure S9b), suggesting that further dilution of IL-2 levels on the nanocapsules could improve the specific targeting of regulatory CD25high T cells. In conclusion, these data indicate that the controlled functionalization of HES nanocapsules with various amounts of IL-2 allows for a dose-dependent targeting of T cells with various IL-2 receptor affinities. 9221

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Figure 6. In vivo uptake of HES-D-IL-2 nanocapsules. In vivo uptake of HES-D-IL-2 and control HES-D nanocapsules by human CD4+CD25+ ̈ immunodeficient Rag2−/−γc−/− mice which T cells was assessed by flow cytometry analysis 4 h after injection of the nanocapsules in naive + + + were reconstituted with human CD4 T cells. (a) Gating strategy of ex vivo human CD4 CD25 T cells. (b) Internalization of HES-D-IL-2 and control HES-D nanocapsules by human CD4+CD25+ T cells (left panel) and uptake of HES-D-IL-2 nanocapsules by CD4+CD25+ vs CD4+CD25low T cells (right panel). The mean percentage ± SD of capsule positive (SR101+) cells are depicted (pooled data of 2 independent experiments). ***p < 0.001.

Results from clinical studies revealed the substantial potential for IL-2 immunotherapy in patients suffering from advanced melanomas or renal cell cancer, resulting in the approval of IL-2 therapy by the FDA.34 High-dose IL-2 induced an efficient stimulation of effector/cytotoxic T cell populations, including CD8+ T cells and NK cells, but was accompanied by severe side effects.14 In contrast, regulatory T cells were the main target of IL-2 low-dose therapy, resulting in activation and proliferation of these T cells.14,17,24,35 Low-dose IL-2 might therefore be suitable as a therapeutic approach for autoimmune and chronic inflammatory disorders. Recently, two clinical trials with patients suffering from systemic lupus erythematosus or type I diabetes have demonstrated that low-dose IL-2 therapy led to a preferential stimulation of regulatory T cells over effector T cells.36,37 Therefore, controlled coupling and assessment of IL-2 amounts on nanocapsules allows the targeting of specific T cell populations with different IL-2 receptor affinities and, therefore, might be highly efficient in immunotherapeutic approaches for both cancer and chronic inflammatory diseases.

(Figure 6). After injection of HES-D-IL-2 or HES-D control nanocapsules, we observed a high percentage of HES-IL-2-D positive human CD4+CD25+ T cells which was significantly enhanced as compared to HES-D control nanocapsules (Figure 6a,b [left panel]). In contrast to CD4+CD25+ human T cells, CD4+CD25low T cells did not show a significant internalization of HED-D-IL-2 nanocapsules (Figure 6b [right panel]). Thus, these data demonstrate a strong CD25-specific uptake of IL-2functionalized nanocapsules by human CD4+CD25+ T cells in vivo. The majority of previous studies used encapsulated IL-2 in nanocarrier-dependent approaches for IL-2 delivery and immune stimulation.28−31 Two studies reported the generation of IL-2 surface conjugation of liposomes32,33 using an Fc framework fused to the C-terminus of the murine IL-2 or covalently bound succinimidyl 4-p-maleimidophenyl butyratemodified IL-2, respectively. Using IL-2 conjugated liposomes, Zheng et al. showed an efficient targeting of murine activated and adoptively transferred CD8 + T cells.37 Here, we successfully have established a reliable protocol for the precise azide-functionalization of IL-2 (Figure S1b-d) and the subsequent nanocapsule modification and quantification of IL-2 (Figure 4a). These techniques allow the concise control and assessment of the cytokine binding, the targeting of T cell populations with different IL-2 receptor affinities (Figure 5), and the analysis of dose-dependent effects of IL-2 on the T cell response (Figure 4). In addition, our study revealed a strong and CD25-specific uptake of HES-D-IL-2 nanocapsules by human CD4+CD25+ in vivo (Figure 6).

CONCLUSIONS Production of biocompatible nanocapsules by miniemulsion process combined with precise cytokine coupling resulted in the generation of IL-2-functionalized HES nanocapsules, which exert potential properties to target human and murine T cells in vitro and in vivo. Here, we established innovative methods to control and to measure the amounts of the nanocapsule bound IL-2, thereby allowing the orchestration of the resulting T cell immune response and the targeting of specific T cell subpopulations with various IL-2 receptor affinities. Direct T 9222

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Generally, the average size and the size distribution of the nanocapsules by means of DLS were measured with diluted dispersions (40 μL sample (solid content 1%) were diluted in 1 mL Ampuwa (or cyclohexane, by using cyclohexane as continuous phase)) on a PSS Nicomp Particle Sizer 380 (Nicomp Particle Sizing Systems, U.S.A.) equipped with a detector at 90 °C scattering mode at 20 °C (Figure 1a). For a more precise measurement of the nanocapsules, the average size and the size distribution were measured on an ALV-5004 multiple-tau full-digital correlator (320 channels) and an ALV spectrometer consisting of a goniometer. This assessment allows measurements over an angular range from 20° to 150° (Figure 1a). A He−Ne laser operating at a laser wavelength of 632.8 nm was used as a light source. For temperaturecontrolled measurements, the light scattering instrument was equipped with a thermostat from Julabo. Diluted dispersions (1 μL sample (solid content 1%) was diluted in 1 mL Ampuwa) were filtered through PTFE membrane filters with a pore size of 5 μm (Chromaphil PES syringe filters). Measurements were performed at 20 °C at 9 angles ranging from 30° to 150°. The zeta potential of the nanocapsules was assessed in 10−3 M potassium chloride solution at 20 °C by use of a Zeta Nanosizer (Malvern Instruments, U.K.) (Figure 1a). Relative fluorescence intensity of the nanocapsules was assessed with a microplate reader Infinite M1000 (Tecan, Germany) (Figure 1a). The encapsulated fluorescent dye SR101 absorbs light at 580 nm and emits light at 605 nm. A total amount of 1 × 1013 nanocapsules·mL−1 (solid content 8 mg) was used for each experiment for data normalization. The experiment was repeated three times. Morphological analysis was conducted by TEM and SEM (Figure 1b). SEM images were achieved by use of a field emission microscope (LEO (Zeiss) 1530 Gemini, Oberkochen, Germany) working at an accelerating voltage of 170 V. In general, the samples were prepared by diluting the nanocapsule dispersion in Ampuwa (for redispersed samples) or cyclohexane to about 0.01% solid content. After a droplet of dispersion was placed onto silica wafers, the samples were dried under ambient conditions overnight. TEM studies were conducted by freeze-drying the nanocapsule dispersion and mixing with EPON. Afterward the EPON samples were cut into 60 nm thick pieces (LEICA Ultramikrotom UCT), stained with ruthenium, and placed on a carbon-coated TEM grid. Images were recorded on a ZEISS 912 microscope working at an accelerating voltage of 120 kV. The solid content of the capsule dispersion was measured gravimetrically. Quantification of Functional Groups on the Nanocapsule Surface. Using the results of the titration experiments performed on a particle charge detector (Mütek GmbH, Germany) combined with a 702 SM Titrino (Automatic Titrator, Metrohm AG, Switzerland), the level of surface charged density was calculated. The amount of NH2 groups present on the capsule surface was measured based on titration experiments on a particle charge detector. To assess the point of zero charge, the amine groups on the nanocapsule surface were titrated against the negatively charged polyelectrolyte poly(ethylene sulfonate) (PES-Na). The titration was performed on 10 mL of the dispersion with a solid content of 1 g·L−1. The amount of charged groups per gram and groups per nanocapsule as calculated from the consumed volume of the polyelectrolyte solution (eqs 1 and 2) were used for the calculations. In eq 1: V is the volume of consumed polyelectrolyte in liters, M is the molar concen-

cell-targeting is an emerging and effective approach in nanoparticle-based immunotherapy not only for cancer but also for inflammatory immune reactions in allergic and autoimmune diseases and transplantation medicine. Therefore, in the long run, delivery of small molecules, drugs, or siRNA by IL-2 nanocapsules should facilitate the modulation of T cell response, resulting in an improved immunotherapy.

METHODS AND MATERIALS Materials. Ampuwa water was used in all experiments. The HES (10%, Mw = 200,000 g·mol−1, degree of molar substitution = 0.5) was purchased from Fresenius Kabi. 2,4-Toluene diisocyanate (TDI) and cyclohexane (>99.9%) were purchased from Sigma-Aldrich. The oil-soluble surfactant poly((ethyleneco-butylene)-b-(ethylene oxide)) P(E/B-b-EO), consisting of a poly(ethylene-co-butylene) block (Mw = 3700 g·mol−1) and a poly(ethylene oxide) block (Mw = 3600 g·mol−1), was synthesized under anionic polymerization conditions.38 The anionic surfactant sodium dodecyl sulfate (SDS) was purchased from Fluka. The fluorescent dye SR101 (Mw = 606.71 g·mol−1) was purchased from BioChemica, Aldrich. The Pierce 660 nm Protein Assay Kit was purchased from Fisher Scientific. Dibenzylcyclooctyne-PEG5-NHS ester (DBCO, Mw = 693.75 g·mol−1, >90% purity), NHS-PEG4 azide (15-azido-4,7,10,13tetraoxa-pentadecanoic acid succinimidyl ester, Mw = 388.37 g· mol−1, >90% purity) was purchased from Jena Bioscience. + + Phosphate buffered saline (DPBS-buffer, -Mg2 , -Ca2 ) was purchased from Life Technologies. The recombinant human IL-2 (amino acid sequence: MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN RWITFCQSII STLT) is a single nonglycosylated protein composed of 134 amino acids and was purchased from Cell Sciences (source E. coli, 15.4 kDa, lyophilized from sterile filtered PBS, pH 7.5, purity >97% by SDS-PAGE and RPHPLC). N,N-Dimethylformamide (Aldrich), chloroform (VWR), dimethyl sulfoxide (Acros Organics), and pyridine (Acros Organics) were of HPLC grade. 9-Chloromethylanthracene (97%) was purchased from Riedel de Haen. Sodium azide (98%) was purchased from SERVA. Methods. Generation and Characterization of IL-2Functionalized Hydroxyethyl Starch Nanocapsules. HES nanocapsules were generated by employing the inverse (water-in-oil) miniemulsion process (Figure S1a).18 For amine functionalization of the nanocapsule surface the crosslinker TDI was added to HES nanocapsules, and amine groups were introduced by the formation of unstable carbamic acid intermediates between the isocyanate groups and water. The amine groups on HES capsules were further functionalized with dibenzylcyclooctyne (DBCO)-PEG5-NHS ester via NHS ester chemistry to introduce reactive alkyne groups on HES-D capsules (Figure S1b). The cytokine IL-2 (CellSciences) was linked to the capsule surface in a multistep process (Figure S1b) by copper-free click reaction between DBCO-functionalized HES (HES-D) and azide-functionalized IL-2 (Figure S1c,d) under mild conditions. At each step of processing HESD-IL-2 capsules, the functionalized nanocapsules were characterized regarding size, size distribution (dynamic light scattering (DLS), PSS Nicomp Particle Sizer 380), morphology (TEM and SEM), and zeta potential (Figure 1a, Figure S7a). 9223

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ACS Nano tration of polyelectrolyte in moles per liter, NA is Avogadro’s constant (6.022 × 1023 mol·L−1), and SC is the solid content of the nanocapsules in gram. In eq 2, using the nanocapsule size determined by DLS, the amount of groups per nanocapsule and per square unit was calculated. Dn is the average number diameter of the nanocapsule, and ρ is the density of polyurethane (1.05·106 g·l−1). groups/g(polymer) =

V ·M ·NA SC

groups/nanocapsule = groups/g(polymer) ·

IL-2-dependent CTLL-2 cells were seeded at 3000 cells/well in 96-well plates. For an internal control of the assay, IL-2 alone (Proleukin (Chiron); CellSciences) was titrated on CTLL-2 cells (data not shown). HES-D-IL-2 and control HES-D nanocapsules were titrated on CTLL-2 cells at concentrations of 0.001, 0.01, 0.1, 1, 10, 12.5, 25, and 75 μg·mL−1 and incubated at 37 °C for 48 h. Proliferation of CTLL-2 cells was assessed by adding [3H]-thymidine to the culture for 16 h and measuring the counts per minute (cpm). HES-D-IL-2 nanocapsule supernatants, obtained after centrifugation post-dialysis, were also titrated on CTLL-2 cells in an equal volume compared to the capsules. ̈ CD4+CD25−, Activated Nanocapsule Uptake by Naive CD4+CD25+, and Regulatory CD4+CD25high Human T Cells. Peripheral blood mononuclear cells (PBMC) were isolated from human buffy coats or leukapheresis via Ficoll separation according to approval of the local ethics committee of Rhineland-Palatine. CD4+ T cells were labeled with antiCD4+ MicroBeads (MACS systems, Miltenyi Biotec, Germany), and isolation was performed according to manufacturer’s instructions gaining a T cell purity of above 95%. For generation of activated CD4+CD25+ T cells, CD4+CD25low T cells were stimulated with 1 μg·mL−1 anti-CD3 mAb (clone OKT3) and 0.5 μg·mL−1 anti-CD28 mAb (BD Pharmingen) at ̈ CD4+ CD25− (CD45RA+CDRO−) T 37 °C for 16 h. Naive cells were isolated from PBMC by depletion of CD14+CD45RO+CD25+ cells and subsequent enrichment of CD4+ T cells using magnetic beads (MACS systems, Miltenyi Biotec). Regulatory CD4+CD25high (FoxP3+) T cells were obtained from PBMC by enrichment of CD25+ cells using antiCD25 + MicroBeads (MACS systems, Miltenyi Biotec) following depletion of CD8+CD14+CD19+ cells with DynalBeads (Thermo Scientific). Subsequently, regulatory T cells were stimulated with 0.5 μg·mL−1 anti-CD3 mAb (clone OKT3) and 0.5 μg·mL−1 anti-CD28 mAb (BD Pharmingen) at 37 °C for 10 h. Purified CD4+ T cells of the three different T cell populations were stained with the proliferation dye CellTrace Violet (Invitrogen, Molecular Probes) at 1 μg· mL−1 per 1 × 107 cells at 37 °C for 20 min. Control marker staining and CD25 expression on CD4+ T cells were confirmed by flow cytometry analysis. CD4+ T cells were treated with nanocapsules at different concentrations (1 [in some experiments], 12.5 [in some experiments], 25, and 75 μg·mL−1) in XVivo20 (LONZA), and cell samples for flow cytometry analysis were taken after 4, 24, 48, and 72 h. In order to exclude that proliferating T cells are more prone to take up nanocapsules, in initial experiments control nanocapsules (HES and HES-D) were additionally applied with soluble IL-2 (50 U·mL−1) on CD4+CD25+ T cells. Untreated and 50 U·mL−1 IL-2-treated CD4+ T cells were taken as control. For gating strategy of nanocapsule positive (SR101+) cells, CD4+ T cells cultured with 50 U·mL−1 IL-2 were taken as control. In order to measure cytotoxicity after nanocapsule treatment, the cells were stained with the Fixable Viability Dye eFluor780 (eBioscience). For assessment of absolute numbers of positive T cells, cells were counted for 45 s with medium flow rate. With regard to uptake analysis of regular HES-D-IL-2, HES-D-IL2/2, and HESD-IL-2/10 nanocapsules, the percentage of positive T cells was normalized to the absolute number of T cells in the 50 U·mL−1 IL-2-treated control group (=100%) to adjust differences in the individual proliferative capacity of T cells obtained from of different donors. The percentage of nanocapsule positive cells and proliferation of T cells was gained by plotting SR101

(1)

ρ ·Dn 3 ·Π 6

(2)

The protein amount of IL-2 after the azidation step (azidation of IL-2 using NHS-ester-PEG4 azide) was quantified using a Pierce 660 nm Protein Kit in 96-well microplates. For every determination, the reaction kit was added to the sample. The measurements were performed at 660 nm (maximum of the dye−metal−protein complex) after 5 min incubation time at 25 °C. All measurements were performed with a TECAN infinite M1000 Plate Reader. The pure IL-2 was used as standard at each measurement (data not shown). High-performance liquid chromatography (HPLC) measurements on azide-functionalized IL-2 were performed by a gradient-system (multistep system) using an Agilent 1200 Series equipped with a quaternary gradient pump and DAD detector coupled with ELSD (Varian 385-LC) (Figure S1d). Furthermore, a reversed-phase column (called MN C4, pore size 300 A, length 250 mm, diameter 4 mm, corn size 5 mm) purchased by Macherey-Nagel was used. The measurements were done at a wavelength of 210 nm at 20 °C with a gradient of acetonitrile with 0.1% TFA and water with 0.1% TFA with a flow rate of 1 mL min−1 (Figure S1d). Alkyne and IL-2 groups were quantitatively determined by covalent linkage of anth-N3 via click chemistry to the capsule surface (Figure S1b). Due to a fluorogenic reaction, the amount of reacted alkyne groups was calculated as previously described.25 For the quantitative calculation of the alkyne groups using Anth-N3, four experiments were performed to measure the fluorescent enhancement:39 (a) Anth-N3/DMSO (50 μL, 2.28 × 1016 molecules, control experiment), (b) HES capsule dispersion/DMSO (control experiment), (c) Anth-N3/ DMSO mixed with HES-NH2 nanaocapsules (ratio 1:1, 2.28 × 1016 molecules of each), and (d) Anth-N3/DMSO mixed with HES-D capsules (ratio 1:1, 2.28 × 1016 molecules of each). For all experiments the samples (b−d) were stirred 24 h at 25 °C. Before measuring the fluorescence enhancement, the samples were purified by repetitive centrifugation (Sigma 3k-30, RCF 3300, 20 min, two times) in order to remove uncoupled AnthN3 molecules and coupling agents and afterward redispersed in demineralized water. Biological Function of Nanocapsule Coupled IL-2. Biological functionality of IL-2 bound to the capsule surface was determined by titrating the functionalized nanocapsules on IL2-dependent murine CTLL-2 cells (Figure 1c). The murine cytotoxic T cell line CTLL-2 was cultured and maintained in RPMI 1640 medium (Lonza) supplemented with 2 mM Lglutamine (PAA Laboratories), 50 μM β-mercaptoethanol (Sigma), and 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories). For maintenance, the cells were additionally cultured with 50 U·mL−1 IL-2 (Proleukin (Chiron), CellSciences). 9224

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ASSOCIATED CONTENT

measurements against CellTrace Violet detection. The division index of IL-2 induced proliferation was computed with the FlowJo proliferation tool. Inhibitory Studies. IL-2 targeted specificity of the nanocapsules was determined by using anti-CD25 antibody basiliximab (Simulect, Novartis). CD4+CD25+ T cell proliferation assays were performed in the presence and absence of 10 μg·mL−1 basiliximab. For this purpose, activated T cells were seeded at 2.5 × 104 cells/well in 96-well plates together with titrating concentrations of HES-D and HES-D-IL-2 either with or without 10 μg·mL−1 basiliximab. Nanocapsule uptake studies were additionally performed with and without 20 μg·mL−1 basiliximab. Flow Cytometry. Flow cytometry was performed using a BD LSR Fortessa, and flow cytometry data were analyzed utilizing FACSDiva (BD Biosciences) and FlowJo (Treestar, Inc.) software. Anti-CD3 and anti-CD28 mAb stimulated CD4+CD25high T cells were stained with anti-CD4-FITC (Beckman Coulter), anti-CD8-FITC (Beckman Coulter), anti-CD25-PE (Miltenyi), and IgG1-FITC (Beckman Coulter) IgG2b-PE isotype (Miltenyi) controls to confirm activation of CD4+CD25high T cells. Nanocapsule uptake was measured as percentage of nanocapsule positive (SR101+) cells 24, 48, and 72 h after nanocapsule addition. Division index of CellTrace Violet positive cells was assessed employing the FlowJo Proliferation tool. The relative division index was calculated as the ratio of the division index of nanocapsule treated CD4+CD25+ T cells to control cells treated with 50 U·mL−1 IL2 alone. Imaging. Confocal laser scanning microscopy was acquired using a LSM 710 NLO (Carl Zeiss) microscope, and images were processed with the software ZEN 2009 (Carl Zeiss). Activated CD4+CD25+ T cells were seeded at 2 × 105 cells/well in 8-well Nunc Lab-Tek chamber slides (Thermo Scientific), and uptake was analyzed 24, 48, and 72 h after nanocapsule treatment. Prior to imaging, the membrane of the cells was stained with CellMask Deep Red Plasma Membrane Stain (Invitrogen, Molecular Probes) at 1 μg·mL−1. Cell nuclei were stained with Hoechst 33342 (Invitrogen) and excited using a 405 nm laser line. The capsule dye Sulforhodamine101 was excited at 586 nm laser line, whereas CellMask Deep Red was excited at 633 nm. In Vivo Application of Nanocapsules. All animal experiments were in accordance with the German Animal Welfare Act (Landesuntersuchungsamt of the state Rhineland-Palatinate, reference no 23 177-07/G 12-1-078, current institutional guidelines) and executed in agreement with the Helsinki convention for the use and care of animals. Immunodeficient Rag2−/−γc−/− mice were reconstituted intraperitoneally (i.p.) with 107 human purified CD4+ T cells. Subsequently, 150 μg nanocapsules dissolved in 150 μL NaCl solution were injected i.p. Mice were CO2 euthanized 4 h after nanocapsule treatment, and cells obtained by peritoneal lavage were taken for flow cytometry analysis. Cells were stained with antihuman CD3FITC (BD Biosciences), antihuman CD4-PE-Cy7 (BD Biosciences), antihuman CD25-APC (Miltenyi), antihuman CD45-AmCyan (BD Biosciences), antimurin-CD45-PacificBlue (BioLegend), and Fixable Viability Dye eFluor780 (eBioscience). Statistical Analysis. Statistical analysis was evaluated using GraphPad Prism (5.0). Statistical significances were calculated with the paired Student t test and two-way ANOVA. P values of 0.05 or less were considered significant.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b07973. Synthesis and functionalization of HES nanocapsules; Synthesis of 9-(azidomethyl)anthracene (anth-N3); In Vivo application of nanocapsules. Figure S1a: IL-2 functionalization of HES nanocapsules in a multistep process: miniemulsion. Figure S1b: IL-2 functionalization of HES nanocapsules in a multistep process. Figure S1c: IL-2 functionalization of HES nanocapsules in a multistep process: MALDI-TOF. Figure S1d: IL-2 functionalization of HES nanocapsules in a multistep process: HPLC. Figure S2: Biological activity of IL-2 on IL-2-dependent murine CTLL-2 cells. Figure S3: Nanocapsule uptake by human CD4+CD25high T cells. Figure S4: Human CD4+CD25high T cell proliferation induced by HES-D-IL-2. Figure S5: Nanocapsule-induced cytotoxicity. Figure S6: Basiliximab did not affect the uptake of control HES-D nanocapsules in human CD4+CD25+ T cells. Figure S7: HES-D-IL-2, HES-D-IL-2/2, and HESD-IL-2/10-induced CTLL-2 proliferation. Figure S8: ̈ CD4+CD25−, Phenotype and uptake of human naive + + activated effector CD4 CD25 , and regulatory CD4+ CD25high T cell populations. Figure S9: Uptake of HES-D-IL-2, HES-D-IL-2/2, and HES-D-IL-2/10 nanocapsules by human naiv̈ e CD4+CD25 −, activated CD4+CD25+, and regulatory CD4+CD25high T cells. Figure S10: In vivo uptake of HES-D-IL-2 nanocapsules (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions §

These authors contributed equally. S.F., M.D., and G.B. performed experiments, analyzed and interpreted data, and wrote the manuscript. F.W. performed the experiments and analyzed the data. V.M. interpreted data and wrote the manuscript. K.L. and K.S. supervised the experimental work, analyzed, and interpreted data and wrote the manuscript. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the German Research Foundation (DFG): STE791/6-1, STE791/9-1 (K.S.), CRC 1066/B6 (K.L., K.S.), TR156/A4/C5 (K.S.) by the German Cancer Aid (110631) (K.S.), by intramural grants (K.S.), and by Max Planck Graduate Center fellowships (S.F., M.P.D.). We thank Katja Klein, Christian Becker, and Biao Kang for excellent technical assistance and Helmut Jonuleit and Jörg Kirberg for providing us with the immunodeficient Rag2−/−γc−/− mice. The work was supported by IMB’s (Johannes Gutenberg-University Mainz, Germany) Flow Cytometry Core Facility and the use of its FACS Aria (INST 247645-1 FUGG) is gratefully acknowledged. 9225

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Signaling by Therapeutic Antibody Basiliximab. J. Immunol. 2010, 184, 1361−1368. (24) Klatzmann, D.; Abbas, A. K. The Promise of Low-Dose Interleukin-2 Therapy for Autoimmune and Inflammatory Diseases. Nat. Rev. Immunol. 2015, 15, 283−294. (25) Baier, G.; Siebert, J. M.; Landfester, K.; Musyanovych, A. Surface Click Reactions on Polymeric Nanocapsules for Versatile Functionalization. Macromolecules 2012, 45, 3419−3427. (26) Xie, F.; Sivakumar, K.; Zeng, Q. B.; Bruckman, M. A.; Hodges, B.; Wang, Q. A Fluorogenic ’Click’ Reaction of Azidoanthracene Derivatives. Tetrahedron 2008, 64, 2906−2914. (27) Sakaguchi, S.; Miyara, M.; Costantino, C. M.; Hafler, D. A. FOXP3+ Regulatory T Cells in the Human Immune System. Nat. Rev. Immunol. 2010, 10, 490−500. (28) Liu, M. Y.; Miao, T. Z.; Zhu, H. X.; Symonds, A. L. J.; Li, L.; Schurich, A.; Maini, M. K.; Zhang, J. M.; Kennedy, P. T. F.; Li, S. L.; Wang, P. IL-2-Engineered Nano-APC Effectively Activates Viral Antigen-Mediated T Cell Responses from Chronic Hepatitis B Virus-Infected Patients. J. Immunol. 2012, 188, 1534−1543. (29) Park, J.; Wrzesinski, S. H.; Stern, E.; Look, M.; Criscione, J.; Ragheb, R.; Jay, S. M.; Demento, S. L.; Agawu, A.; Limon, P. L.; Ferrandino, A. F.; Gonzalez, D.; Habermann, A.; Flavell, R. A.; Fahmy, T. M. Combination Delivery of TGF-beta Inhibitor and IL-2 by Nanoscale Liposomal Polymeric Gels Enhances Tumour Immunotherapy. Nat. Mater. 2012, 11, 895−905. (30) Fadel, T. R.; Sharp, F. A.; Vudattu, N.; Ragheb, R.; Garyu, J.; Kim, D.; Hong, E.; Li, N.; Haller, G. L.; Pfefferle, L. D.; Justesen, S.; Herold, K. C.; Fahmy, T. M. A Carbon Nanotube-Polymer Composite for T-Cell Therapy. Nat. Nanotechnol. 2014, 9, 639−647. (31) McHugh, M. D.; Park, J.; Uhrich, R.; Gao, W.; Horwitz, D. A.; Fahmy, T. M. Paracrine Co-Delivery of TGF-beta and IL-2 using CD4-Targeted Nanoparticles for Induction and Maintenance of Regulatory T Cells. Biomaterials 2015, 59, 172−181. (32) Konigsberg, P. J.; Godtel, R.; Kissel, T.; Richer, L. L. The Development of IL-2 Conjugated Liposomes for Therapeutic Purposes. Biochim. Biophys. Acta, Biomembr. 1998, 1370, 243−251. (33) Zheng, Y.; Stephan, M. T.; Gai, S. A.; Abraham, W.; Shearer, A.; Irvine, D. J. In Vivo Targeting of Adoptively Transferred T-Cells with Antibody- and Cytokine-Conjugated Liposomes. J. Controlled Release 2013, 172, 426−435. (34) Rosenberg, S. A. IL-2: the First Effective Immunotherapy for Human Cancer. J. Immunol. 2014, 192, 5451−5458. (35) Arenas-Ramirez, N.; Woytschak, J.; Boyman, O. Interleukin-2: Biology, Design and Application. Trends Immunol. 2015, 36, 763−777. (36) Yu, A.; Snowhite, I.; Vendrame, F.; Rosenzwajg, M.; Klatzmann, D.; Pugliese, A.; Malek, T. R. Selective IL-2 Responsiveness of Regulatory T Cells through Multiple Intrinsic Mechanisms Supports the Use of Low-Dose IL-2 Therapy in Type 1 Diabetes. Diabetes 2015, 64, 2172−2183. (37) von Spee-Mayer, C.; Siegert, E.; Abdirama, D.; Rose, A.; Klaus, A.; Alexander, T.; Enghard, P.; Sawitzki, B.; Hiepe, F.; Radbruch, A.; Burmester, G. R.; Riemekasten, G.; Humrich, J. Y. Low-Dose Interleukin-2 Selectively Corrects Regulatory T Cell Defects in Patients with Systemic Lupus Erythematosus. Ann. Rheum. Dis. 2016, 75, 1407−1415. (38) Schlaad, H.; Kukula, H.; Rudloff, J.; Below, I. Synthesis of Alpha,omega-heterobifunctional Poly(ethylene glycol)s by Metal-Free Anionic Ring-Opening Polymerization. Macromolecules 2001, 34, 4302−4304. (39) Wang, X. L.; Xie, S. G.; Zhang, L.; Yang, W. X.; Wang, X.; Jin, H. Z. Comparison of Ligase Detection Reaction and Real-Time PCR for Detection of Low Abundant YMDD Mutants in Patients with Chronic Hepatitis B. World J. Gastroenterol. 2008, 14, 120−124.

REFERENCES (1) Steinman, R. M.; Banchereau, J. Taking Dendritic Cells into Medicine. Nature 2007, 449, 419−426. (2) Paulis, L. E.; Mandal, S.; Kreutz, M.; Figdor, C. G. Dendritic CellBased Nanovaccines for Cancer Immunotherapy. Curr. Opin. Immunol. 2013, 25, 389−395. (3) Tang, Q.; Bluestone, J. A. The Foxp3+ Regulatory T Cell: a Jack of all Trades, Master of Regulation. Nat. Immunol. 2008, 9, 239−244. (4) Chaput, N.; Conforti, R.; Viaud, S.; Spatz, A.; Zitvogel, L. The Janus Face of Dendritic Cells in Cancer. Oncogene 2008, 27, 5920− 5931. (5) Priyadharshini, B.; Greiner, D. L.; Brehm, M. A. T-Cell Activation and Transplantation Tolerance. Transplant. Rev. 2012, 26, 212−22. (6) Islam, S. A.; Luster, A. D. T Cell Homing to Epithelial Barriers in Allergic Disease. Nat. Med. 2012, 18, 705−715. (7) Blazar, B. R.; Murphy, W. J.; Abedi, M. Advances in Graft-VersusHost Disease Biology and Therapy. Nat. Rev. Immunol. 2012, 12, 443− 458. (8) Bluestone, J. A.; Bour-Jordan, H.; Cheng, M.; Anderson, M. T Cells in the Control of Organ-Specific Autoimmunity. J. Clin. Invest. 2015, 125, 2250−2260. (9) Steward-Tharp, S. M.; Song, Y. J.; Siegel, R. M.; O’Shea, J. J. New Insights Into T Cell Biology and T Cell-Directed Therapy for Autoimmunity, Inflammation, and Immunosuppression. Ann. N. Y. Acad. Sci. 2010, 1183, 123−148. (10) Serra, P.; Santamaria, P. Nanoparticle-Based Autoimmune Disease Therapy. Clin. Immunol. 2015, 160, 3−13. (11) Shao, K.; Singha, S.; Clemente-Casares, X.; Tsai, S.; Yang, Y.; Santamaria, P. Nanoparticle-Based Immunotherapy for Cancer. ACS Nano 2015, 9, 16−30. (12) Lowenthal, J. W.; MacDonald, H. R.; Iacopetta, B. J. Intracellular Pathway of Interleukin 2 Following Receptor-Mediated Endocytosis. Eur. J. Immunol. 1986, 16, 1461−1463. (13) Malek, T. R. The Biology of Interleukin-2. Annu. Rev. Immunol. 2008, 26, 453−479. (14) Boyman, O.; Sprent, J. The Role of Interleukin-2 during Homeostasis and Activation of the Immune System. Nat. Rev. Immunol. 2012, 12, 180−190. (15) Saadoun, D.; Rosenzwajg, M.; Joly, F.; Six, A.; Carrat, F.; Thibault, V.; Sene, D.; Cacoub, P.; Klatzmann, D. Regulatory T-Cell Responses to Low-Dose Interleukin-2 in HCV-Induced Vasculitis. N. Engl. J. Med. 2011, 365, 2067−2077. (16) Yu, A.; Zhu, L.; Altman, N. H.; Malek, T. R. A Low Interleukin2 Receptor Signaling Threshold Supports the Development and Homeostasis of T Regulatory Cells. Immunity 2009, 30, 204−217. (17) Malek, T. R.; Castro, I. Interleukin-2 Receptor Signaling: at the Interface Between Tolerance and Immunity. Immunity 2010, 33, 153− 165. (18) Baier, G.; Baumann, D.; Siebert, J. M.; Musyanovych, A.; Mailander, V.; Landfester, K. Suppressing Unspecific Cell Uptake for Targeted Delivery using Hydroxyethyl Starch Nanocapsules. Biomacromolecules 2012, 13, 2704−2715. (19) Kolb, H. C.; Sharpless, K. B. The Growing Impact of Click Chemistry on Drug Discovery. Drug Discovery Today 2003, 8, 1128− 1137. (20) Duprez, V.; Ferrer, M.; Cornet, V.; Olive, D.; Dautry-Varsat, A. Modulation of Interleukin 2 Internalization and Interleukin 2Dependent Cell Growth by Antireceptor Antibodies. J. Biol. Chem. 1991, 266, 1497−1501. (21) Lamaze, C.; Dujeancourt, A.; Baba, T.; Lo, C. G.; Benmerah, A.; Dautry-Varsat, A. Interleukin 2 Receptors and Detergent-Resistant Membrane Domains Define a Clathrin-Independent Endocytic Pathway. Mol. Cell 2001, 7, 661−671. (22) Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. SizeDependent Internalization of Particles via the Pathways of Clathrinand Caveolae-Mediated Endocytosis. Biochem. J. 2004, 377, 159−169. (23) Du, J. M.; Yang, H.; Zhang, D. P.; Wang, J. C.; Guo, H. Z.; Peng, B. Z.; Guo, Y. J.; Ding, J. P. Structural Basis for the Blockage of IL-2 9226

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