Combining Adhesive Nanostructured Surfaces and Costimulatory

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Cite This: Nano Lett. XXXX, XXX, XXX−XXX

Combining Adhesive Nanostructured Surfaces and Costimulatory Signals to Increase T Cell Activation Judith Guasch,*,†,‡ Marco Hoffmann,§,∥ Jennifer Diemer,§,∥ Hossein Riahinezhad,§,∥,# Stefanie Neubauer,⊥ Horst Kessler,⊥ and Joachim P. Spatz*,§,∥

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†

Dynamic Biomaterials for Cancer Immunotherapy, Max Planck Partner Group, Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus UAB, E-08193 Bellaterra, Spain ‡ Department of Molecular Nanoscience and Organic Materials, ICMAB-CSIC and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Campus UAB, E-08193 Bellaterra, Spain § Department of Cellular Biophysics, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany ∥ Department of Biophysical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, D-69120 Heidelberg, Germany ⊥ Institute for Advanced Study (IAS) and Center of Integrated Protein Science (CIPSM), Department Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany S Supporting Information *

ABSTRACT: Adoptive cell therapies are showing very promising results in the fight against cancer. However, these therapies are expensive and technically challenging in part due to the need of a large number of specific T cells, which must be activated and expanded in vitro. Here we describe a method to activate primary human T cells using a combination of nanostructured surfaces functionalized with the stimulating anti-CD3 antibody and the peptidic sequence arginine-glycine-aspartic acid, as well as costimulatory agents (anti-CD28 antibody and a cocktail of phorbol 12-myristate 13-acetate, ionomycin, and protein transport inhibitors). Thus, we propose a method that combines nanotechnology with cell biology procedures to efficiently produce T cells in the laboratory, challenging the current state-of-the-art expansion methodologies. KEYWORDS: Nanostructures, PMA/ionomycin, CD3/CD28, T cells, integrins

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strength of the signal provided by the nanostructures to activate T cells was clearly insufficient. In this study, we present three modifications of the process that greatly improve the results obtaining proliferation rates comparable (and even higher) than Dynabeads.17,18 First, the concentration of the stimulating ligands, which determines the rate of T cell triggering, was increased. Second, the contribution of costimulatory molecules to improve the amplification of the signal was enhanced. Third, the duration of the interaction between the TCR and the stimulating nanostructures was extended in order to increase the signal accumulation. Specifically, a prestimulatory step with phorbol 12-myristate 13-acetate (PMA)/ionomycin (and the protein transport inhibitors brefeldin A and monensin that promote the accumulation of secreted proteins) was added. This triggers interleukin 2 (IL-2) synthesis and the expression of its receptor through the activation of protein kinase C (PKC) and an increase of the cytosolic-free calcium concentration.19−24 Moreover, the protein-repellent poly(ethylene glycol) (PEG)

doptive T cell therapy represents a promising treatment for many cancer patients, especially those with an advanced disease.1−3 However, this methodology, which is based on the administration of ex vivo manipulated T cells to patients, still faces several challenges before it can be implemented on a large scale.4−6 One of these challenges is to obtain a fast and affordable expansion of primary human T cells in vitro.4,5 An effective T cell activation and proliferation requires T cell receptor (TCR) ligation7 and costimulatory signals, which include membrane receptors of the antigen presenting cells such as CD288−10 and the presence of cytokines from inflammatory, homeostatic, or autocrine sources.11 Functionalized nanostructured surfaces produced by block copolymer micellar lithography (BCML) are a new method to stimulate T cells ex vivo, which affords precise control at the nanoscale.12−15 Specifically, the ligand density can be easily tuned through the distance between the nanoparticles (NPs) that decorate the surfaces, whereas the ligand orientation can be controlled by choosing an appropriate functionalization strategy.16 Nevertheless, the activation and proliferation rates obtained so far were about 30 times smaller than the state-of-the-art commercial Dynabeads (Thermo Fisher Scientific, U.S.A.). Thus, the © XXXX American Chemical Society

Received: June 26, 2018 Revised: July 27, 2018

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DOI: 10.1021/acs.nanolett.8b02588 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 1. (a) Scanning electron microscopy (SEM) image of a TiO2 substrate (dark gray background) decorated with AuNPs (white dots) separated 34 ± 7 nm, (b) fluorescence image of a substrate half of which decorated with AuNPs by dip-coating and functionalized with a fluorescently marked aCD3, (c) fluorescence image of rat embryonic fibroblast (REF) cells that were seeded on functionalized substrates overnight (blue, nuclei; green, actin filaments stained with phalloidin; red, focal adhesions stained with the antibody anti-paxillin).

and the anti-CD3 (aCD3) or anti-CD28 (aCD28) antibodies on the AuNPs. RGD is known to be a binding site of the extracellular matrix (ECM) protein fibronectin for different integrins, such as α5β1, which stimulates cell adhesion.33 This molecule, which contained phosphonic acids as anchoring groups due to their stability under aqueous conditions once anchored onto TiO2,34,35 was used to functionalize the interparticle areas in order to enhance the cell−substrate interaction36,37 and prevent unspecific protein adsorption to TiO2. To immobilize aCD3 on the AuNPs, a thiolated nitrilotriacetic acid molecule was coordinated to a his-tagged protein G through niquel (II) ions, which served as an anchoring point for the antibody. To analyze the results, we prepared substrates that were decorated by dip-coating with AuNPs on only half of their surface, resulting in a nanostructured area and a non-nanostructured one, separated by the so-called dipping line. By functionalizing the whole substrate with a fluorescently marked aCD3, fluorescence was only observed on the nanostructured part, confirming that the antibody was specifically immobilized on the AuNPs (Figure 1b). Additionally, the low fluorescence of the area above the dipping line also indicates that the unspecific adsorption of antibodies was residual. In the next step, rat embryonic fibroblasts (REF) cells were seeded on the nanostructures to evaluate the cell-adhesive properties of the RGD-functionalized TiO2. The comparable cell area values obtained when using the functionalized nanostructured substrates (4270 ± 1106 μm2) and the standard culture dishes (4216 ± 1387 μm2) as well as the presence of actin filaments and focal adhesions (Figure 1c) confirmed the cell-adhesive properties of the surfaces. After functionalizing the nanostructured substrates, primary human CD4+ T cells were purified from blood of healthy adult donors and seeded on functionalized nanostructured surfaces. Additionally, we performed a prestimulatory step in some experiments, consisting of incubating the cells in standard medium supplemented with a costimulatory cocktail of PMA, ionomycin, brefeldin A, and monensin. The combination of PMA and ionomycin triggers IL-2 synthesis and the expression of its receptor, through the activation of PKC and an increase of the cytosolic-free calcium concentration, while the protein inhibitors promote the accumulation of secreted proteins.19−24 Moreover, soluble aCD28 was added in chosen samples because of its role as a costimulatory agent through its binding to the receptor CD28 on the surface of T cells.8−10 The obtained results were compared to those of Dynabeads (microbeads coated with aCD3 and aCD28), which served

previously employed was substituted with a cell-adhesive background, consisting of the tripeptide arginine-glycineaspartic acid (RGD), which should enhance the duration of the T cell−nanostructure.15,25,26 More specifically, the cyclic RGDfK derivative was chosen,27 which activates different integrins such as α5β1 (IC50 = 141 ± 15 nM).28 This integrin is expressed by T cells and has been related to enhanced proliferation.29,30 Finally, nanostructures with various interparticle distances, hence with various concentrations of functional ligands available to the cells, were used.14,15 With the objective of obtaining large amounts of CD4+ T cells in the laboratory, we first prepared nanostructured surfaces consisting of quasi-hexagonally ordered gold nanoparticles (AuNPs) on TiO2 surfaces by BCML following a protocol described previously.31,32 Briefly, block copolymers consisting of polystyrene and 2-polyvinylpyridine, PS(x)-bP2VP(y) were dissolved in an apolar solvent to form micelles consisting of a hydrophobic PS shell and a hydrophilic P2VP core. Then, gold(III) chloride trihydrate was dissolved into the polar micellar cores, where it loses a proton that interacts with the nitrogen atom of the P2VP chain. Thus, the loaded micelles are stabilized by electrostatic interactions created between the positively charged NH+ of the polymer and the negatively charged AuCl4−. The interparticle distance can be varied by using different polymer lengths, whereas the size of the AuNPs can be tuned by changing the loading of the micelles (Table S1). To coat the TiO2 surfaces, which were previously produced by sputtering glass slides with TiO2, two techniques were employed, dip- and spin-coating. In dipcoating, substrates are immersed into the micellar solution and pulled out at a constant speed, forming a two-dimensional compact quasi-hexagonally structured monolayer of loaded micelles on TiO2, due to capillary forces during the evaporation of the solvent. The interparticle distance can also be tuned by changing the velocity of the dip-coating process. In the spin-coating process, a precise volume of micellar solution is deposited onto the substrate, which is rotating at a high speed to thin the fluid and dry the excess of solvent from the resulting film. In this case, the distance between AuNPs can be modified by varying the acceleration of the rotating support, the final speed, and the rotation time. To produce the nanostructures, the coated substrates were first plasma treated and then heated in an oven to high temperature to reinforce the anchoring of the particles, thus avoiding particle removal by the cells (Figure 1a). We then proceeded with their functionalization, which included a cell-adhesive RGD background on the TiO2 layer B

DOI: 10.1021/acs.nanolett.8b02588 Nano Lett. XXXX, XXX, XXX−XXX

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Figure 2. (a) IL-2 secretion of prestimulated CD4+ T cells with PMA/ionomycin (plus protein transport inhibitors) using different activation methods (Ndonors = 8 with a minimum of Ndonors/condition = 2): (1) Soluble aCD28 and nanostructured surfaces consisting of an RGD-functionalized TiO2 substrate decorated with immobilized aCD3 on the AuNPs separated about 35 nm (aCD3i/aCD28s), nanostructured substrates consisting of an RGD-functionalized TiO2 layer decorated with (2) aCD3 or (3) aCD28 immobilized on the AuNPs separated about 35 nm (aCD28i); (4) Dynabeads, positive control; (5) dish, no further stimulation was applied, negative control. (b) IL-2 secretion of prestimulated (blue boxes) and untreated (black boxes) CD4+ T cells (Ndonors = 8 with a minimum of Ndonors/condition = 5) obtained using soluble aCD28 and nanostructured surfaces consisting of an RGD-functionalized TiO2 substrate decorated with immobilized aCD3 on the AuNPs separated x nm (x = 35, 60, 100, and 150 nm). The results of prestimulated CD4+ T cells with aCD3i/aCD28s and Dynabeads shown in (a) is shown again in (b) for comparison. Statistical significance was determined by the Mann−Whitney test (* p < 0.5, ** p < 0.1, *** p < 0.05).

in our experiments is similar (about 95 mm2/culture well) to the total surface of the nanostructures (about 80 mm2/culture well), thus ensuring their comparability. To evaluate the effect of the prestimulatory step as well as the density of ligands, the IL-2 secretion of prestimulated and untreated CD4+ T cells obtained using soluble aCD28 and nanostructured surfaces consisting of an RGD-functionalized TiO2 substrate decorated with aCD3 immobilized on the AuNPs separated x nm (x = 35, 60, 100, and 150) was analyzed (Figure 2b). The positive control Dynabeads was also evaluated. As expected, the absence of the prestimulatory step with the stimulatory cocktail gave considerably less IL-2 secretion when cells were cultured on functionalized nanostructured substrates with all median values smaller than 1 ng/ mL (Figure S1). In contrast, the IL-2 secretion using Dynabeads only decreased from 25.6 ng/mL when prestimulated to the 19.8 ng/mL obtained without the costimulatory cocktail, thus resulting in the most efficient activation system when PMA/ionomycin and the protein transport inhibitors are not used. Nevertheless, prestimulated CD4+ T cells showed the highest IL-2 secretion on nanostructured substrates with the smallest interparticle distance (35 nm) (38.7 ng/mL) as mentioned above, although the median obtained is not significantly different than the one obtained with substrates decorated with AuNPs separated 60 nm (37.5 ng/mL). Moreover, cells seeded on nanostructures with the largest interparticle distance (150 nm) still show higher IL-2 concentrations (35.1 ng/mL) than Dynabeads, even though the differences were only significant for the substrates with AuNPs separated 100 nm (37.8 ng/mL). Additionally, the expression of the CD69 membrane receptor was evaluated, which is known to be an early activation marker due to its transient upregulation when the T cell receptor is activated and the T cells remain in the secondary lymphoid organs,41

as positive control, given that they are considered the state-ofthe-art commercial polyclonal expansion method. Initially, five different activation methods, all of which included the prestimulation with the costimulatory cocktail, were compared by assessing the IL-2 secretion through an enzyme linked immunosorbent assay (ELISA) 16−20 h after cell seeding (Figure 2a). The first method consisted of soluble aCD28 combined with nanostructured substrates consisting of an RGD-functionalized TiO2 layer decorated with aCD3 immobilized on AuNPs separated ca. 35 nm (aCD3i/aCD28s). In the second, we eliminated the soluble aCD28 (aCD3i/-), whereas in third one we substituted the immobilized aCD3 with immobilized aCD28 (aCD28i/-). The fourth method was the positive control, that is, the Dynabeads, while the fifth was the negative control, which consisted only of prestimulation with the cocktail. Prestimulated CD4+ T cells activated on nanostructured substrates with immobilized aCD3 and soluble aCD28 showed the highest IL-2 secretion with a median of 38.7 ng/mL, exceeding the secretion obtained with the stateof-the-art commercial Dynabeads (median = 25.6 ng/mL) and with the nanostructures functionalized with only aCD28 (median = 27.3 ng/mL). The aCD3i/- sample resulted in a lower IL-2 secretion with a median of 19.4 ng/mL, suggesting that the addition of aCD3 to a stimulating system that contains PMA/ionomycin (17.8 ng/mL) does not result in a significant improvement, as previously reported.38 Conversely, the addition of aCD28 gives higher IL-2 secretion as demonstrated in both the aCD28i/and Dynabead systems. Surprisingly, the combination of soluble aCD28 and immobilized aCD3 on the nanostructures resulted in the highest activation value. Under these conditions, the different stimulation inputs seem to result in a positive effect,39 which could be caused by partially different signaling pathways of aCD3 and PMA/ionomycin.40 It is worth mentioning that the total surface area of Dynabeads C

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Figure 3. (a) Proliferation rates of prestimulated CD4+ T cells on day 7 (Ndonors = 8 with a minimum of Ndonors/condition = 6) and (b) percentages of CD45RO+ and CD45RA+ prestimulated CD4+ T cells on day 6 (Ndonors = 8, with a minimum of Ndonors/condition = 6). Statistical significance was determined by the Mann−Whitney test (ns, no difference; * p < 0.5).

automating the production process by using, for example, a high-throughput approach.42,43 Thus, these experiments could result, for example, in a slightly larger variability of the activation of the aCD28i/- sample or reduce the variability between approaches observed in the proliferation analysis. Finally, the phenotypes of the expanded CD4+ T cells were assessed by determining the percentages of CD45RA+ and CD45RO+ T cells44 on day 6 by flow cytometry (Figure 3b), given their relevance for T cell functionality45 and clinical outcome.46 Indeed, specific T cell subsets, for example, the central memory T cell subset, which is CD45RA+, have been shown to be beneficial for the treatment of certain diseases such as cancer due to their high proliferative potential in vivo. CD4+ T cells expanded on RGD/aCD3-functionalized nanostructures with interparticle distances of about 35 nm costimulated with soluble aCD28 produced slightly more CD45RA+ (34.4%) and less CD45RO+ (81.2%) T cells than Dynabeads, which yielded percentages of 21.2% and 89.5%, respectively. In summary, we described a method for activating primary human CD4+ T cells in vitro using the combination of a prestimulation step, functionalized nanostructured surfaces, and costimulatory compounds. The nanostructured surfaces consisting of covalently functionalized RGD on rigid TiO2 surfaces decorated with quasi-hexagonal arrays of AuNPs linked to the stimulating antibody aCD3, enhanced cell− substrate contact through the RGD, while polyclonally activating the TCR complex through aCD3. In addition, the prestimulatory step with the cocktail of PMA, ionomycin, and protein transport inhibitors stimulated the cells by generating

confirming the results obtained by analyzing the secretion of the cytokine IL-2 (Figure S2). Next, we analyzed the proliferation rates obtained 7 days after seeding the prestimulated CD4+ T cells on RGD/aCD3functionalized nanostructures with interparticle distances of about 35 nm costimulated with soluble aCD28. For that, CD4+ T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and analyzed by flow cytometry (Figures 3a and S3). The expansion index, which determines the fold-expansion of the whole population, was higher for cells expanded on functionalized nanostructured substrates costimulated with soluble aCD28 (8.9) than for Dynabeads (6.7). Similarly, the division index, that is, the average number of cell divisions that a cell from the original population has undergone (including those cells which did not divide), was also higher for cells expanded on nanostructures (2.0) than for those expanded using Dynabeads (1.9), thus confirming that the activation results shown in Figure 2 resulted in successful proliferations. Nevertheless, the translation of such proof-of-concept results to the clinics would require the evaluation of the system on a larger set of donors to explore the intrinsic variability of the primary samples. Thus, parameters such as the age of the donor, which might affect both proliferation and differentiation, as well as others directly related to the disease (e.g., low white blood cell counts, availability of tumor-specific lymphocytes, etc.) and comorbidities should be evaluated. Additionally, the effect of possible defects related to the functionalized nanostructured surfaces, which might have remained despite sample replication, would also be minimized. Moreover, this aspect could be further improved by D

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regulatory intracellular signals such as the activation of PKC, which provides a calcium influx into the T cell, resulting in an improved IL-2 secretion of about 2 orders of magnitude when using the nanostructured surfaces. Moreover, cells that were further costimulated with soluble aCD28, which prevents anergy by binding to the B7 complexes, showed doubled IL-2 secretion than cells that were not. We therefore demonstrated that the combination of certain signals has an additive effect in promoting cell activation and proliferation, in agreement with a previous report,39 which could be exploited to improve the available lymphocyte expansion systems.

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REFERENCES

(1) Kalos, M.; Levine, B. L.; Porter, D. L.; Katz, S.; Grupp, S. A.; Bagg, A.; June, C. H. T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia. Sci. Transl. Med. 2011, 3, 95ra73. (2) Porter, D. L.; Levine, B. L.; Kalos, M.; Bagg, A.; June, C. H. Chimeric Antigen Receptor−Modified T Cells in Chronic Lymphoid Leukemia. N. Engl. J. Med. 2011, 365, 725−733. (3) Porter, D. L.; Hwang, W.-T.; Frey, N. V.; Lacey, S. F.; Shaw, P. A.; Loren, A. W.; Bagg, A.; Marcucci, K. T.; Shen, A.; Gonzalez, V.; Ambrose, D.; Grupp, S. A.; Chew, A.; Zheng, Z.; Milone, M. C.; Levine, B. L.; Melenhorst, J. J.; June, C. H. Chimeric Antigen Receptor T Cells Persist and Induce Sustained Remissions in Relapsed Refractory Chronic Lymphocytic Leukemia. Sci. Transl. Med. 2015, 7, 303ra139. (4) Crompton, J. G.; Sukumar, M.; Restifo, N. P. Uncoupling T-Cell Expansion from Effector Differentiation in Cell-Based Immunotherapy. Immunol. Rev. 2014, 257, 264−276. (5) Butler, M. O.; Hirano, N. Human Cell-Based Artificial AntigenPresenting Cells for Cancer Immunotherapy. Immunol. Rev. 2014, 257, 191−209. (6) Pérez del Río, E.; Martinez Miguel, M.; Veciana, J.; Ratera, I.; Guasch, J. Artificial 3D Culture Systems for T Cell Expansion. ACS Omega 2018, 3, 5273−5280. (7) Zehn, D.; Lee, S. Y.; Bevan, M. J. Complete but Curtailed T-Cell Response to Very Low-Affinity Antigen. Nature 2009, 458, 211−214. (8) June, C. H.; Ledbetter, J. A.; Linsley, P. S.; Thompson, C. B. Role of the CD28 Receptor in T-Cell Activation. Immunol. Today 1990, 11, 211−216. (9) Koulova, L.; Clark, E. A.; Shu, G.; Dupont, B. The CD28 Ligand B7/BB1 Provides Costimulatory Signal for Alloactivation of CD4+ T Cells. J. Exp. Med. 1991, 173, 759−762. (10) Levine, B. L.; Bernstein, W. B.; Connors, M.; Craighead, N.; Lindsten, T.; Thompson, C. B.; June, C. H. Effects of CD28 Costimulation on Long-Term Proliferation of CD4+ T Cells in the Absence of Exogeneous Feeder Cells. J. Immunol. 1997, 159, 5921− 5930. (11) Curtsinger, J. M.; Schmidt, C. S.; Mondino, A.; Lins, D. C.; Kedl, R. M.; Jenkins, M. K.; Mescher, M. F. Inflammatory Cytokines Provide a Third Signal for Activation of Naive CD4+ and CD8+ T Cells. J. Immunol. 1999, 162, 3256−3262. (12) Delcassian, D.; Depoil, D.; Rudnicka, D.; Liu, M.; Davis, D. M.; Dustin, M. L.; Dunlop, I. E. Nanoscale Ligand Spacing Influences Receptor Triggering in T Cells and NK Cells. Nano Lett. 2013, 13, 5608−5614. (13) Deeg, J.; Axmann, M.; Matic, J.; Liapis, A.; Depoil, D.; Afrose, J.; Curado, S.; Dustin, M. L.; Spatz, J. P. T Cell Activation Is Determined by the Number of Presented Antigens. Nano Lett. 2013, 13, 5619−5626. (14) Matic, J.; Deeg, J.; Scheffold, A.; Goldstein, I.; Spatz, J. P. Fine Tuning and Efficient T Cell Activation with Stimulatory aCD3 Nanoarrays. Nano Lett. 2013, 13, 5090−5097. (15) Guasch, J.; Muth, C. A.; Diemer, J.; Riahinezhad, H.; Spatz, J. P. Integrin-Assisted T-Cell Activation on Nanostructured Hydrogels. Nano Lett. 2017, 17, 6110−6116. (16) Groll, J.; Albrecht, K.; Gasteier, P.; Riethmueller, S.; Ziener, U.; Moeller, M. Nanostructured Ordering of Fluorescent Markers and Single Proteins on Substrates. ChemBioChem 2005, 6, 1782−1787. (17) van Panhuys, N. TCR Signal Strength Alters T-DC Activation and Interaction Times and Directs the Outcome of Differentiation. Front. Immunol. 2016, 7, 1−14. (18) Lanzavecchia, A.; Sallusto, F. Progressive Differentiation and Selection of the Fittest in the Immune Response. Nat. Rev. Immunol. 2002, 2, 982−987. (19) Chatila, T.; Silverman, L.; Miller, R.; Geha, R. Mechanisms of T Cell Activation by the Calcium Ionophore Ionomycin. J. Immunol. 1989, 143, 1283−1289. (20) Berry, N.; Nishizuka, Y. Protein Kinase C and T Cell Activation. Eur. J. Biochem. 1990, 189, 205−214.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b02588. Materials and methods, IL-2 secretion of non-prestimulated CD4+ T cells, CD69 expression of CD4+ T cells,

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CFSE profiles of CD4+ T cells (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Judith Guasch: 0000-0002-3571-4711 Horst Kessler: 0000-0002-7292-9789 Present Address #

(H.R.) Department of Chemical Engineering, Queen’s University, 19 Division St., K7L 3N6 Kingston, Canada.

Author Contributions

J.G. designed the experiments and concept, performed experiments, and wrote the manuscript; M.H., J.D., and H.R. supported the experiments; S.N. and H.K. provided the RGD molecule; J.P.S. designed the experiments and concept and supported manuscript writing; all authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We kindly acknowledge M. Kern and U. Sentner for fruitful discussions as well as D. P. Rosenblatt for proofreading the manuscript. The work was supported by the People programme (Marie Curie Actions) of the 7th Framework Programme of the European Union (FP7/2007-2013) through an Intra-European fellowship (PIEF-GA-2012-329908) as well as the Grant Agreement Nr. 600388 of REA with the Agency for Business Competitiveness ACCIÓ (Tecniospring fellowship, TECSPR15-1-0015). We are also grateful to the Max Planck Society, especially for the funding of the Max Planck Partner Group “Dynamic Biomimetics for Cancer Immunotherapy” in collaboration with the Max Planck Institute for Medical Research. ICMAB acknowledges support from the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2015-0496). E

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Nano Letters (21) Hashimoto, S.; Takahashi, Y.; Tomita, Y.; Hayama, T.; Sawada, S.; Horie, T.; McCombs, C. C.; Michalski, J. P. Mechanism of Calcium Ionophore and Phorbol Ester-Induced T-Cell Activation. Accessory Cell Requirement for T-Cell Activation. Scand. J. Immunol. 1991, 33, 393−403. (22) Pelham, H. R. B. Multiple Targets for Brefeldin A. Cell 1991, 67, 449−451. (23) Klausner, R. D.; Donaldson, J. G.; Lippincott-Schwartz, J. Brefeldin A: Insights into the Control of Membrane Traffic and Organelle Structure. J. Cell Biol. 1992, 116, 1071−1080. (24) Mollenhauer, H. H.; Morré, D. J.; Rowe, L. D. Alteration of Intracellular Traffic by Monensin; Mechanism, Specificity and Relationship to Toxicity. Biochim. Biophys. Acta, Rev. Biomembr. 1990, 1031, 225−246. (25) Cavalcanti-Adam, E. A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J. P. Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands. Biophys. J. 2007, 92, 2964−2974. (26) Guasch, J.; Diemer, J.; Riahinezhad, H.; Neubauer, S.; Kessler, H.; Spatz, J. P. Synthesis of Binary Nanopatterns on Hydrogels for Initiating Cellular Responses. Chem. Mater. 2016, 28, 1806−1815. (27) Haubner, R.; Gratias, R.; Diefenbach, B.; Goodman, S. L.; Jonczyk, A.; Kessler, H. Structural and Functional Aspects of RGDContaining Cyclic Pentapeptides as Highly Potent and Selective Integrin αvβ3 Antagonists. J. Am. Chem. Soc. 1996, 118, 7461−7472. (28) Kapp, T. G.; Rechenmacher, F.; Neubauer, S.; Maltsev, O. V.; Cavalcanti-Adam, E. A.; Zarka, R.; Reuning, U.; Notni, J.; Wester, H.J.; Mas-Moruno, C.; Spatz, J.; Geiger, B.; Kessler, H. A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-Binding Integrins. Sci. Rep. 2017, 7, 39805. (29) Davis, L. S.; Oppenheimer-Marks, N.; Bednarczyk, J. L.; McIntyre, B. W.; Lipsky, P. E. Fibronectin Promotes Proliferation of Naive and Memory T Cells by Signaling through Both the VLA-4 and VLA-5 Integrin Molecules. J. Immunol. 1990, 145, 785−793. (30) Neto, E. H.; Coelho, A. L. J.; Sampaio, A. L. F.; Henriques, M. d. G. M. O.; Marcinkiewicz, C.; De Freitas, M. S.; Barja-Fidalgo, C. Activation of Human T Lymphocytes Via Integrin Signaling Induced by RGD-Disintegrins. Biochim. Biophys. Acta, Mol. Cell Res. 2007, 1773, 176−184. (31) Spatz, J. P.; Moessmer, S.; Hartmann, C.; Moeller, M.; Herzog, T.; Krieger, M.; Boyen, H.-G.; Ziemann, P.; Kabius, B. Ordered Deposition of Inorganic Clusters from Micellar Block Copolymer Films. Langmuir 2000, 16, 407−415. (32) Lohmueller, T.; Aydin, D.; Schwieder, M.; Morhard, C.; Louban, I.; Pacholski, C.; Spatz, J. P. Nanopatterning by Block Copolymer Micelle Nanolithography and Bioinspired Applications. Biointerphases 2011, 6, MR1−MR12. (33) Pierschbacher, M. D.; Ruoslahti, E. Cell Attachment Activity of Fibronectin Can Be Duplicated by Small Synthetic Fragments of the Molecule. Nature 1984, 309, 30−33. (34) Rechenmacher, F.; Neubauer, S.; Mas-Moruno, C.; Dorfner, P. M.; Polleux, J.; Guasch, J.; Conings, B.; Boyen, H.-G.; Bochen, A.; Sobahi, T. R.; Burgkart, R.; Spatz, J. P.; Fässler, R.; Kessler, H. A Molecular Toolkit to Functionalize Ti-Based Biomaterials That Selectively Control Integrin-Mediated Cell Adhesion. Chem. - Eur. J. 2013, 19, 9218−9223. (35) Guasch, J.; Conings, B.; Neubauer, S.; Rechenmacher, F.; Ende, K.; Rolli, C. G.; Kappel, C.; Schaufler, V.; Micoulet, A.; Kessler, H.; Boyen, H.-G.; Cavalcanti-Adam, E. A.; Spatz, J. P. Segregation Versus Colocalization: Orthogonally Functionalized Binary Micropatterned Substrates Regulate the Molecular Distribution in Focal Adhesions. Adv. Mater. 2015, 27, 3737−3747. (36) Hu, B.; Leow, W. R.; Cai, P.; Li, Y.-Q.; Wu, Y.-L.; Chen, X. Nanomechanical Force Mapping of Restricted Cell-to-Cell Collisions Oscillating between Contraction and Relaxation. ACS Nano 2017, 11, 12302−12310. (37) Hu, B.; Leow, W. R.; Amini, S.; Nai, B.; Zhang, X.; Liu, Z.; Cai, P.; Li, Z.; Wu, Y.-L.; Miserez, A.; Lim, C. T.; Chen, X. Orientational

Coupling Locally Orchestrates a Cell Migration Pattern for ReEpithelialization. Adv. Mater. 2017, 29, 1700145. (38) Weiss, A.; Imboden, J. B. Cell Surface Molecules and Early Events Involved in Human T Lymphocyte Activation. Adv. Immunol. 1987, 41, 1−38. (39) Marchingo, J. M.; Kan, A.; Sutherland, R. M.; Duffy, K. R.; Wellard, C. J.; Belz, G. T.; Lew, A. M.; Dowling, M. R.; Heinzel, S.; Hodgkin, P. D. Antigen Affinity, Costimulation, and Cytokine Inputs Sum Linearly to Amplify T Cell Expansion. Science 2014, 346, 1123− 1127. (40) Li, M. D.; McAllen, K.; Sharp, B. M. Regulation of Delta Opioid Receptor Expression by Anti-CD3-ε, PMA, and Ionomycin in Murine Splenocytes and T Cells. J. Leukocyte Biol. 1999, 65, 707−714. (41) Shiow, L. R.; Rosen, D. B.; Brdickova, N.; Xu, Y.; An, J.; Lanier, L. L.; Cyster, J. G.; Matloubian, M. CD69 Acts Downstream of Interferon-α/β to Inhibit S1P1 and Lymphocyte Egress from Lymphoid Organs. Nature 2006, 440, 540−546. (42) Cai, P.; Zhang, X.; Wang, M.; Wu, Y.-L.; Chen, X. Combinatorial Nano−Bio Interfaces. ACS Nano 2018, 12, 5078− 5084. (43) Cai, P.; Leow, W. R.; Wang, X.; Wu, Y.-L.; Chen, X. Programmable Nano−Bio Interfaces for Functional Biointegrated Devices. Adv. Mater. 2017, 29, 1605529. (44) Bujan, W.; Schandene, L.; Ferster, A.; De Valck, C.; Goldman, M.; Sariban, E. Abnormal T-Cell Phenotype in Familial Erythrophagocytic Lymphohistiocytosis. Lancet 1993, 342, 1296−1298. (45) Zhu, J.; Yamane, H.; Paul, W. E. Differentiation of Effector CD4 T Cell Populations. Annu. Rev. Immunol. 2010, 28, 445−489. (46) Hinrichs, C. S.; Borman, Z. A.; Gattinoni, L.; Yu, Z.; Burns, W. R.; Huang, J.; Klebanoff, C. A.; Johnson, L. A.; Kerkar, S. P.; Yang, S.; Muranski, P.; Palmer, D. C.; Scott, C. D.; Morgan, R. A.; Robbins, P. F.; Rosenberg, S. A.; Restifo, N. P. Human Effector CD8+ T Cells Derived from Naive Rather Than Memory Subsets Possess Superior Traits for Adoptive Immunotherapy. Blood 2011, 117, 808−814.

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DOI: 10.1021/acs.nanolett.8b02588 Nano Lett. XXXX, XXX, XXX−XXX