Letter pubs.acs.org/NanoLett
Integrin-Assisted T‑Cell Activation on Nanostructured Hydrogels Judith Guasch,*,†,‡,§,∥ Christine A. Muth,§,∥ Jennifer Diemer,§,∥ Hossein Riahinezhad,§,∥ and Joachim P. Spatz§,∥ †
Dynamic Biomaterials for Cancer Immunotherapy, Max Planck Partner Group, Institute of Materials Science of Barcelona (ICMAB-CSIC), Campus UAB, Bellaterra, E-08193, Spain ‡ Department of Molecular Nanoscience and Organic Materials, Institute of Materials Science of Barcelona (ICMAB-CSIC) and Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Campus UAB, Bellaterra, E-08193, Spain § Department of Cellular Biophysics, Max Planck Institute for Medical Research, Jahnstrasse 29, Heidelberg, D-69120, Germany ∥ Department of Biophysical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, Heidelberg, D-69120, Germany S Supporting Information *
ABSTRACT: Adoptive cell therapy (ACT) has shown very promising results as treatment for cancer in a few clinical trials, such as the complete remissions of otherwise terminal leukemia patients. Nevertheless, the introduction of ACT into clinics requires overcoming not only medical but also technical challenges, such as the ex vivo expansion of large amounts of specific T-cells. Nanostructured surfaces represent a novel T-cell stimulation technique that enables us to fine-tune the density and orientation of activating molecules presented to the cells. In this work, we studied the influence of integrin-mediated cell-adhesion on T-cell activation, proliferation, and differentiation using nanostructured surfaces, which provide a well-defined system at the nanoscale compared with standard cultures. Specifically, we synthesized a polymeric polyethylene glycol (PEG) hydrogel cross-linked with two fibronectin-derived peptides, cyclic Arg-Gly-Asp (cRGD) and cyclic Leu-Asp-Val (cLDV), that are known to activate different integrins. Moreover, the hydrogels were decorated with a quasi-hexagonal array of gold nanoparticles (AuNPs) functionalized with the activating antibody CD3 to initiate T-cell activation. Both cLDV and cRGD hydrogels showed higher T-cell activation (CD69 expression and IL-2 secretion) than nonfunctionalized PEG hydrogels. However, only the cRGD hydrogels clearly supported proliferation giving a higher proportion of cells with memory (CD4+CD45RO+) than naı̈ve (CD4+CD45RA+) phenotypes when interparticle distances smaller than 150 nm were used. Thus, T-cell proliferation can be enhanced by the activation of integrins through the RGD sequence. KEYWORDS: Nanostructures, hydrogels, T-cells, peptides, integrins the integrin αLβ2, also named lymphocyte function-associated antigen 1 (LFA-1), of T-cells.14 Other receptors responsible for co-stimulatory signals are also present in the IS, such as the protein CD28 that binds to CD80 and CD86 and boosts T-cell activation.15−18 The formation of the IS results in the reorganization of the cytoskeleton through the orientation of actin filaments and microtubules toward the IS, which might facilitate clustering and prevent T-cell migration.19,20 In vitro, TCRs can be polyclonally activated with an anti-CD3 antibody (aCD3), an invariable component of the TCR that bypasses antigen recognition and initiates signal transmission.21,22 However, the presence of a co-stimulatory signal is necessary to obtain complete T-cell activation, which is usually given by an anti-CD28 antibody (aCD28) and/or ICAM-1.18,23,24 In
T
he immune system protects the body against pathogens and eliminates dead cells but shows difficulties in recognizing and destroying cancer cells, probably due to their self-origin.1−4 Recently, it has been shown that is possible to artificially assist the immune system using adoptive cell therapy (ACT), which is based on the extraction of T-cells from patients, their ex vivo treatment, and reintroduction to the patients.5−7 To obtain satisfactory results, large amounts of functional T-cells must be produced in a short period of time, which is a challenging task with the current technologies.8,9 The development of more efficient culture systems to activate and expand functional T-cells is therefore highly desirable.10,11 In vivo, T-cells are activated by antigen presenting cells (APCs) through the immunological synapse (IS).12 The IS is a dynamic cell interface that consist of the T-cell receptor (TCR) and an agonist peptide, the antigen, presented through a protein complex, the major histocompatibility complex (MHC), by the APCs.13 To sustain this contact, the intercellular adhesion molecule 1 (ICAM-1) is presented on APCs and interacts with © XXXX American Chemical Society
Received: June 22, 2017 Revised: August 25, 2017
A
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
Figure 1. (a) Box plots summarizing the CD69 expression and IL-2 secretion of primary human CD4+ T-cells (Ndonors = 5, with a minimum of Ndonors/condition = 5) activated by commercial Dynabeads (Thermo Fisher Scientific) seeded on noncoated and fibronectin-coated dishes. The boxes correspond to the interquartile range (defined by the 25th and 75th percentiles), the central line is the median, the whiskers show one standard deviation, and the square is the average. (b) Photographs of a peptide-containing PEG hydrogel with a ruler (scale in centimeters) and SEM image showing its cLDV background (gray) decorated with AuNPs (white dots, interparticle distance = 69 ± 11 nm), scale bar = 200 nm (inset). (c) Scheme of the components and polymerization reaction that result in peptide-containing nanostructured PEG hydrogels. (d) Fluorescence image of the area around the dipping line of a cLDV hydrogel decorated with Alexa Fluor 488-tagged aCD3-functionalized AuNPs (scale bar = 25 μm). (e) Fluorescence image of REF52 cells that remained on a cRGD hydrogel decorated with aCD3-functionalized AuNPs 12 h after seeding (blue, nuclei; green, actin filaments stained with phalloidin; red, focal adhesions stained with antipaxillin) (scale bar = 100 μm). Statistical significance was determined by the Mann−Whitney U test (ns: no significant difference).
there is a relapse at a later stage). Thus, memory and effector cells have fewer co-stimulatory requirements than naı̈ve cells.27 These differences are due to the expression of various cell adhesion molecules (CAMs), such as integrins. For example, the receptors α4β1, α4β7, and LFA-1 are highly expressed on effector T-cells, while naı̈ve T-cells only show low levels.27 Moreover, integrins bind not only cellular receptors of the APCs and ECs but also different extracellular matrix (ECM) proteins that are present in the secondary lymphoid organs such as collagen, laminin, and fibronectin.29,30 Specifically, fibronectin has been observed to co-stimulate T-cell proliferation by signaling through the integrins α4β1 and α5β1.31,32
addition to the IS, T-cells form another type of dynamic synapse with endothelial cells (ECs).13 This interaction determines T-cell homing, recirculation, and extravasation to the inflamed sites.25,26 Such processes depend on the surface phenotype of cells, which varies between T-cell subsets.27 Effector cells exhibit a high capacity for adherence, which is necessary to fulfill their function of localizing at the sites of inflammation, as opposed to inactivated naı̈ve cells, which circulate in the blood.27 Memory cells, which are generated during the immunological response, largely retain the surface phenotype and migratory characteristics of effector cells28 and act as effector cells during a second immune response (i.e., if B
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters α4β1 recognizes the Leu-Asp-Val (LDV) sequence, located in the V region of fibronectin, which is known to activate LFA-1.33 Initially, LFA-1 binds weakly to ICAM-1 and only after recognition of the TCR or a cytokine receptor will a conformation change take place, which results in cell proliferation and differentiation. On the other hand, the binding site for α5β1 is the well-known sequence Arg-GlyAsp (RGD),34 which was the first sequence identified to be bound by integrins35 and interacts with ca. one-third of all human integrins.36 Nevertheless, the influence of such minimal structures, consisting only of short peptides, on T-cell activation remains largely unexplored at the nanoscale level. Nanostructured surfaces consisting of glass decorated with quasi-hexagonal arrays of AuNPs produced by block copolymer micellar lithography (BCML) have recently been used to stimulate T-cells ex vivo.37−39 Such nanostructured surfaces enable us to determine ligand spacing and density at the nanoscale, thus controlling distances that can influence receptor−ligand interactions and receptor clustering.40 This control is achieved by varying the copolymer length used in the BCML as well as the coating parameters. Briefly, the BCML consists of dissolving an amphiphilic block copolymer in an apolar solvent and loading the created reverse micelles with a metallic precursor. Rigid substrates are then coated with the loaded micelles, resulting in nanostructured surfaces after a plasma treatment. Additionally, such surfaces can be functionalized with ligands, whose orientation can also be wellestablished by specific functionalization protocols.41 Nanostructured surfaces are therefore excellent platforms with which to study the influence of ligands on cells at the nanoscale in an extremely well-defined environment, which is difficult to obtain with other culturing techniques. With this objective, Matic el al.39 polyclonally stimulated primary human CD4+ T-cells using arrays of AuNPs functionalized with aCD3 surrounded by glass passivated with a PEG monolayer. A correlation between the aCD3 density and the T-cell activation response was observed. Nevertheless, the effect of CAMs was never assessed in this platform designed to analyze the minimal requirements for Tcell activation. Here, we go one step further in complexity and analyzed the influence of fibronectin-mediated cell adhesion of primary human CD4+ T-cell activation, proliferation, and differentiation using cell-adhesive hydrogels decorated with TCR-stimulating nanoarrays. Specifically, AuNPs were functionalized with aCD3, whereas PEG hydrogels were crosslinked with cyclic Arg-Gly-Asp (cRGD) and cyclic Leu-Asp-Val (cLDV). The peptide-containing PEG hydrogels were preferred over coated glass to avoid unspecific cell adhesion. Such a process can arise on modified glass due to the detachment of the passivation layer under aqueous conditions.42 We therefore present a robust platform that enables us to study the influence of specific cell adhesion interactions on T-cell activation in a well-controlled system that successfully eliminates any potential overshadowing effect. Initially, the importance of the ECM protein fibronectin on T-cell activation was investigated. Primary human CD4+ T-cells purified from peripheral blood (purity CD3+CD4+ T-cells >90%) were polyclonally activated with commercial beads coated with aCD3 and aCD28 (Dynabeads, Thermo Fisher Scientific), the state-of-the-art T-cell expansion technology, on noncoated dishes and dishes covered with fibronectin (Figure 1a). To evaluate the results, two early activation markers were measured after 16−20 h of incubation by flow cytometry and an enzyme-linked immunosorbent assay (ELISA), the CD69
expression and the IL-2 secretion, respectively. CD69 is transiently up-regulated by activated TCRs, preventing the expression of the sphingosine-1-phopsphate receptor (S1PR1) at the cell surface.43 Consequently, CD69 increases during the period that recently activated T-cells remain in the secondary lymphoid organs. Similarly, the cytokine IL-2 is secreted rapidly by activated T-cells and is known to support T-cell proliferation and differentiation by interaction with the IL-2 receptor. It is used in clinics to evaluate lymphocyte function. Figure 1a shows increased CD69 expression and IL-2 secretion when fibronectin was used in comparison with noncoated dishes, suggesting a co-stimulatory effect of fibronectin on T-cell activation. Thus, we decided to thoroughly explore the role of its two minimal binding sites RGD and LDV, which are believed to be responsible for enhanced proliferation. We therefore included such peptides in our synthetic nanostructured surfaces. These surfaces consisted of peptidecontaining PEG hydrogels decorated with quasi-hexagonal arrays of AuNPs (Figure 1b). The synthesis of such hydrogels was performed through the combination of a previously described co-polymerization process to produce nanostructured hydrogels44 with a thiol− acrylate coupling reaction (Figure 1c). The addition of thiyl radicals to carbon−carbon double bonds is a click chemistry reaction due to its high efficiency, high reaction rates, and mild conditions required including insensitivity to oxygen and water.45,46 Additionally, cross-linked polymers obtained through a thiol−ene photopolymerization process result in very homogeneous networks, showing narrow glass transition regions and low polymerization shrinkage stress.46−48 In this case, an aqueous solution of a thiolated peptide, cRGD or cLDV, was added to acrylate PEG macromers. The mixture was spread on glass slides decorated with quasi-hexagonal arrays of acrylate functionalized AuNPs, which were co-polymerized via a photoinduced UV reaction in the presence of an initiator with the peptides and PEG macromers. Thus, peptide-containing PEG hydrogels decorated with AuNPs were synthesized. As we previously described,39 such AuNPs were functionalized with aCD3. Briefly, aCD3 were immobilized on protein G through their Fc part, resulting in a preferred orientation for cell recognition. The anchoring of protein G to AuNPs was achieved by the formation of a coordination complex between the six histidine tag of protein G, a nitrilotriacetic acid (NTA) that was directly linked to the AuNPs through its thiol group, and a nickel divalent ion. This functionalization strategy should result in one protein G per AuNP,37,39 which can support ca. 5 aCD3 per protein G, given the multiple binding sites of the protein.49 Thus, the aCD3 density of the surface is proportional to the AuNP density. To ensure compatibility between the cRGD and cLDV peptides and the aCD3 functionalization protocol, we prepared hydrogels that were nanostructured on only half of the available surface. Specifically, substrates were decorated with AuNPs spaced 35 nm apart using the dipcoating method so that a border (dipping line) between the coated and the noncoated areas was obtained. By the incubation of the whole hydrogels with aCD3 labeled with Alexa Fluor 488, fluorescence was only observed on the area below the dipping line, i.e., the area coated with AuNPs (Figures 1d and S1). This result confirms that aCD3 was successfully immobilized on the AuNPs. Moreover, the low fluorescence of the area above the dipping line indicates that the interaction between aCD3 and the peptides is minimal. Finally, it is also worth mentioning that the thicker lines above C
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
Figure 2. (a) Box plots summarizing the percentage of CD69 expression (left) and IL-2 secretion (right) of primary human CD4+ T-cells activated by PEG hydrogels containing no peptide (dark yellow lines), cRGD (green lines), and cLDV (orange lines) decorated with aCD3-functionalized AuNPs separated by 35 nm and co-stimulated with soluble aCD28. (b) Box plots summarizing the percentage of CD69 expression on cRGD and cLDV hydrogels decorated with aCD3-functionalized AuNPs separated by various distances (20, 35, 60, 100, and 150 nm) and co-stimulated with soluble aCD28. “Blank” samples are peptide-containing hydrogels (cRGD or cLDV) without nanostructures, whereas “35 nm w/o aCD28” are samples consisting of aCD3-functionalized AuNPs separated 35 nm that were not co-stimulated with aC28. (c) Box plots summarizing the IL-2 secretion of the samples described in panel b. The results shown were obtained from Ndonors = 17, with a minimum of Ndonors/condition = 3. The boxes correspond to the interquartile range (defined by the 25th and 75th percentiles), the central line is the median, the whiskers show one standard deviation, and the square is the average. Statistical significance was determined by the Mann−Whitney U test (ns: no significant difference; *, p < 0.5; **, p < 0.05; ***, p < 0.005).
with our previously described nanostructured PEG-passivated glass substrates decorated with aCD3.39 This difference could be explained by the unspecific binding of cells on partially decomposed PEG passivation layers. This decomposition could have arisen from the instability of silanes used to anchor the PEG chains on the glass surfaces under aqueous conditions.42 After analyzing the importance of including a peptidic sequence on PEG hydrogels decorated with aCD3-functionalized AuNPs separated 35 nm, the influence of the interparticle distance was assessed. For that, the results depicted in Figure 2a for peptide-containing PEG hydrogels were compared with their analogous hydrogels with interparticle distances ranging from 20 to 150 nm. On cRGD-containing hydrogels, cells expressed CD69 when the AuNPs were not separated more than 100 nm, whereas hydrogels containing cLDV required higher amounts of aCD3 (Figure 2b). Specifically, interparticle distances of 60 nm were necessary. Above these thresholds, the CD69 expression on cLDV hydrogels was constant. A similar behavior was observed for hydrogels with cRGD, although there was a small increase for nanostructures with interparticle distances of 35 nm in comparison with the substrates that presented AuNPs separated 60 nm. Moreover, cRGD hydrogels were always significantly more efficient than the cLDV ones. Similarly, the IL-2 secretion of CD4+ T-cells was also higher for hydrogels containing cRGD than for cLDV hydrogels (Figure 2c). The thresholds observed for IL-2 secretion were similar to the ones obtained for CD69 expression. Nevertheless, the IL-2
the dipping line are accumulations of AuNPs, which always emerge near to the dipping line as an intrinsic artifact of the dip-coating process. In addition, we also demonstrated the functionality of the peptides by seeding rat embryonic fibroblasts (REF52) on the hydrogels and studying their cell adhesion. Cells only spread on the RGD-containing hydrogels (Figures 1e and S1) as predicted, due to the absence of cell adhesive sequences on the other hydrogels for REF52 cells. Therefore, we further confirmed that the peptides remained functional after AuNP functionalization. After demonstrating the successful surface functionalization using dip-coated surfaces, we prepared hydrogels by spincoating to ensure the complete coverage of the surface of the hydrogels with AuNPs so that cells could only contact nanostructured areas. Figure 2 shows the CD69 expression and IL-2 secretion of primary human CD4+ T-cells seeded on aCD3-functionalized nanostructured hydrogels and costimulated with soluble aCD28. The green boxes correspond to cells seeded on cRGD-containing PEG hydrogels, whereas the orange boxes correspond to cells seeded on PEG hydrogels cross-linked with cLDV. Additionally, Figure 2a displays dark yellow boxes, which show the results of PEG hydrogels that were not cross-linked to any peptidic sequence. As observed for fibronectin-coated dishes, higher CD69 expression levels and IL-2 secretion were obtained for aCD3-functionalized hydrogels that can trigger integrins. In fact, the values obtained using peptide-free hydrogels were practically negligible in contrast D
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
Figure 3. Box plots summarizing the percentage of divided CD4+ T-cells obtained on cRGD- (green lines) and cLDV- (orange lines) containing PEG hydrogels decorated with aCD3-functionalized AuNPs separated by various distances (20, 35, 60, 100, and 150 nm) and co-stimulated with soluble aCD28 on days (a) 3 and (c) 7. Representative CFSE-profiles of divided CD4+ T-cells on cRGD hydrogels (green lines) on days (b) 3 and (d) 7 for 20, 35, and 150 nm and blank samples. The dark gray peak on the left corresponds to cells that were not stained with CFSE, whereas the light gray peak on the right is given by undivided CFSE-stained cells. Box plots summarizing the percentage of (e) CD4+CD45RA+ (naı̈ve phenotype) and (f) CD4+CD45RO+ (memory phenotype) CD4+ T-cells obtained on the nanostructured cRGD (green lines) and cLDV (orange lines) hydrogels described in panels a−d on day 6. “Blank” samples are peptide-containing PEG hydrogels (cRGD or cLDV) without nanostructures, whereas “35 nm w/o aCD28” are samples consisting of aCD3-functionalized AuNPs separated 35 nm that were not co-stimulated with soluble aC28. The results shown were obtained from Ndonors = 9, with a minimum of Ndonors/condition = 2. The boxes correspond to the interquartile range (defined by the 25th and 75th percentiles), the central line is the median, the whiskers show one standard deviation, and the square is the average. Statistical significance was determined by the Mann−Whitney U test (ns: no significant difference; *, p < 0.5; **, p < 0.1; ***, p < 0.05).
After the evaluation of the short-term activation, CD4+ T-
secretion obtained with cRGD hydrogels with interparticle distances of 20 nm was higher than the secretion measured on 35 nm cRGD hydrogels. It is worth noting that cells seeded on both cRGD and cLDV hydrogels expressed CD69 without CD28 stimulation but did not produce significant IL-2, as previously described for passivated glass substrates.39
cells were stained with carboxyfluorescein succinimidyl ester (CFSE) to evaluate their proliferation rates.50 The percentage of divided cells was calculated for the different samples on days 3 (Figure 3a) and 7 (Figure 3c). E
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
RGD activates different vitronectin-related integrins as well as α5β1, which are associated with cell adhesion and proliferation.32,54−56 Accordingly, our nanostructured surfaces showed higher T-cell activation and proliferation rates on cell adhesive substrates than on cell repellent environments such as pure PEG. Moreover, cRGD hydrogels presented higher activation rates than cLDV hydrogels. Specifically, interparticle distances of 100 nm were sufficient to induce IL-2 secretion and CD69 expression in cRGD hydrogels, whereas cLDV hydrogels required interparticle distances of 60 nm. Thus, less aCD3 was necessary to activate CD4+ T-cells seeded on cRGD than on cLDV hydrogels. More importantly, only cRGD hydrogels were capable to induce proliferation. Few non-exclusive reasons could explain this behavior such as the enhancement of the Tcell−nanostructure interaction due to a strong cell-adhesion capacity of RGD, resulting in an increase of the IS signal strength, the stimulation of α5β1-related signaling pathways by RGD, which are known to enhance cell proliferation,57,58 or potentially overlapping functions of LDV and the costimulatory aCD28.59 It is also worth mentioning that CD4+ T-cells showed similar activation values when using cRGD hydrogels or the previously reported PEG-passivated glass, both decorated with quasi-hexagonal nanoarrays functionalized with aCD3. In the latter, unspecific cell−substrate interactions probably arise from the detachment of unstable PEG from glass surfaces and its substitution by proteins of the media. In conclusion, these results suggest that enhanced T-cell activation and expansion could be obtained by mimicking some interactions of T-cells with proteins of the ECM. Specifically, the addition of the commercially available and affordable RGD sequence to the marketed aCD3/aCD28 stimulation microbead systems could improve them. Such a sequence could be even included using nanostructures to further optimize its amount and orientation in combination with antibodies using, for example, nanostructures consisting of more than one type of NP.60
Cells seeded on nanostructured hydrogels that were not cross-linked to any peptidic sequence did not show proliferation, in agreement with the lack of IL-2 secretion on day 1. Similarly, cLDV hydrogels did not promote cell expansion on days 3 (Figure 3a, oranges boxes) and 7 (Figure 3c, orange boxes) in accordance with their low IL-2 secretion. In contrast, Figure 3b,d shows a representative example of the CFSE profiles obtained on days 3 and 7 by flow cytometry when cells were seeded on cRGD hydrogels (green lines), respectively. The x-axis shows the fluorescence intensity, which decreases to the left side, while the y-axis depicts the relative cell counts, with the peaks representing the divisions of the cells. The dark gray peak on the left correspond to cells that were not stained with CFSE, demonstrating that they are not autofluorescent. The light gray peak on the right represents undivided CFSE-cells. All of the functionalized nanostructured samples show peaks (green lines) with lower fluorescence than the light gray peak, indicating that cells divided. Specifically, cells divided ca. 3−4 times on day 3, which increased to 7−8 times on day 7, before the CFSE becomes undetectable. As observed with the CD69 expression and IL-2 secretion, cells proliferate on nanostructures with interparticle distances smaller than 100 nm, reaching ca. 30% of divided cells on day 7 with the smallest interparticle distance. The phenotype of the obtained cells was also determined given its dependence on TCR signal strength, which is determined by the concentration of aCD3 and costimulatory molecules as well as the duration of the T-cell−artificial APC interaction.51,52 On day 6, the distribution of CD4+CD45RA+ (naı̈ve phenotype) and CD4+CD45RO+ (memory phenotype) expanded T-cells53 was measured as shown in panels e and f of Figure 3, respectively. Not surprisingly, the more the cells proliferate, the higher the percentage of cells obtained with a memory phenotype and the lower the percentage of cells obtained with a naı̈ve phenotype. Specifically, ca. 90% of the cells expanded on the 35 nm cRGD hydrogels were CD4+CD45RO+ T-cells. Finally, it is worth mentioning that the variation observed between donors is believed to be mainly caused by the intrinsic variability among primary samples. Nevertheless, defects on the nanostructures and their functionalization cannot be completely discarded in spite of sample replication. To reduce such variation in view of a potential clinical use, the establishment of certain donor (patient) subsets as well as the automation of the hydrogel fabrication should be explored. Additionally, the increased number of experiments needed to bridge the gap between this proof-of-concept and a potential clinical use might also reduce sample variability. In this work, we analyzed the importance of fibronectinmediated cell adhesion for T-cell activation through a nanostructured system designed to study the minimal conditions for large and rapid T-cell expansions, as required in adoptive T-cell therapies. Specifically, we employed quasihexagonal structures of AuNPs with interparticle distances of 20 to 150 nm supported by PEG hydrogels. These hydrogels were cross-linked with the peptides RGD or LDV, two integrin bindings sites of fibronectin that have been related to T-cell proliferation through the integrins α5β1 and α4β1, respectively.31,32 Moreover, α4β1 and other integrins such as α4β7 that are crucial for T-cell function, enabling for example leukocyte extravasation,26 interact with LDV. Additionally, the characterized sites within ligands that bind the β2 integrins such as LFA-1, whose ligation prevents T-cell anergy after TCR engagement, are structurally similar to LDV.33 In contrast,
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b02636. Additional details on materials and methods. Figures showing molecular structures of cRGD and cLDV, surface functionalization strategy, and CD4+ T-cell clustering on day 6. A table showing preparation parameters. (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Judith Guasch: 0000-0002-3571-4711 Joachim P. Spatz: 0000-0003-3419-9807 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We acknowledge D. P. Rosenblatt for proofreading this paper. This work was funded by the European Union Seventh Framework Program through a Marie Curie IEF (PIEF-GAF
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
(34) Johansson, S.; Svineng, G.; Wennerberg, K.; Armulik, A.; Lohikangas, L. Front. Biosci., Landmark Ed. 1997, 2, 126−146. (35) Pierschbacher, M. D.; Ruoslahti, E. Nature 1984, 309, 30−33. (36) Zent, R.; Pozzi, A. Cell-Extracellular Matrix Interactions in Cancer; Springer: New York, USA, 2010. (37) Deeg, J.; Axmann, M.; Matic, J.; Liapis, A.; Depoil, D.; Afrose, J.; Curado, S.; Dustin, M. L.; Spatz, J. P. Nano Lett. 2013, 13, 5619−5626. (38) Delcassian, D.; Depoil, D.; Rudnicka, D.; Liu, M.; Davis, D. M.; Dustin, M. L.; Dunlop, I. E. Nano Lett. 2013, 13, 5608−5614. (39) Matic, J.; Deeg, J.; Scheffold, A.; Goldstein, I.; Spatz, J. P. Nano Lett. 2013, 13, 5090−5097. (40) Cavalcanti-Adam, E. A.; Volberg, T.; Micoulet, A.; Kessler, H.; Geiger, B.; Spatz, J. P. Biophys. J. 2007, 92, 2964−2974. (41) Groll, J.; Albrecht, K.; Gasteier, P.; Riethmueller, S.; Ziener, U.; Moeller, M. ChemBioChem 2005, 6, 1782−1787. (42) Arkles, B.; Pan, Y.; Larson, G. L.; Singh, M. Chem. Eur. J. 2014, 20, 9442−9450. (43) Shiow, L. R.; Rosen, D. B.; Brdickova, N.; Xu, Y.; An, J.; Lanier, L. L.; Cyster, J. G.; Matloubian, M. Nature 2006, 440, 540−546. (44) Aydin, D.; Louban, I.; Perschmann, N.; Bluemmel, J.; Lohmueller, T.; Cavalcanti-Adam, E. A.; Haas, T. L.; Walczak, H.; Kessler, H.; Fiammengo, R.; Spatz, J. P. Langmuir 2010, 26, 15472− 15480. (45) Dénès, F.; Pichowicz, M.; Povie, G.; Renaud, P. Chem. Rev. 2014, 114, 2587−2693. (46) Hoyle, C. E.; Bowman, C. N. Angew. Chem., Int. Ed. 2010, 49, 1540−1573. (47) Hoyle, C. E.; Lee, T. Y.; Roper, T. J. Polym. Sci. A 2004, 42, 5301−5338. (48) Jacobine, A. F., In Radiation Curing in Polymer Science Technology III, Fouassier, J. D.; Rabek, J. F., Eds; Elsevier: London, UK, 1993; pp 219−268. (49) Guss, B.; Eliasson, M.; Olsson, A.; Uhlen, M.; Frej, A.-K.; Jornvall, H.; Flock, J.-I.; Lindberg, M. EMBO J. 1986, 5, 1567−1575. (50) Lyons, A. B.; Parish, C. R. J. Immunol. Methods 1994, 171, 131− 137. (51) Lanzavecchia, A.; Sallusto, F. Nat. Rev. Immunol. 2002, 2, 982− 987. (52) Iezzi, G.; Karjalainen, K.; Lanzavecchia, A. Immunity 1998, 8, 89−95. (53) Mackay, C. R. Nature 1999, 401, 659−660. (54) D’Souza, S. E.; Ginsberg, M. H.; Plow, E. F. Trends Biochem. Sci. 1991, 16, 246−250. (55) 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. Adv. Mater. 2015, 27, 3737−3747. (56) 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. Chem. Eur. J. 2013, 19, 9218−9223. (57) Schwartz, M. A.; Assoian, R. K. J. Cell Sci. 2001, 114, 2553− 2560. (58) Moreno-Layseca, P.; Streuli, C. H. Matrix Biol. 2014, 34, 144− 153. (59) Kohlmeier, J. E.; Rumsey, L. M.; Chan, M. A.; Benedict, S. H. Immunology 2003, 108, 152−157. (60) Guasch, J.; Diemer, J.; Riahinezhad, H.; Neubauer, S.; Kessler, H.; Spatz, J. P. Chem. Mater. 2016, 28, 1806−1815.
2012-329908) and a Marie Curie Co-fund project (TECSPR151-0015).
■
REFERENCES
(1) Houghton, A. N. J. Exp. Med. 1994, 180, 1−4. (2) Bubanovic, I. Origin of Anti-Tumor Immunity Failure in Mammals; Springer Science + Business Media, Inc.: New York, USA, 2004. (3) Alexander, M. A. Immune-based Cancer Treatment - The T lymphocyte Response; CRC Press - Taylor & Francis Group: Boca Raton, FL, USA, 2011. (4) Houghton, A. N.; Guevara-Patiño, J. A. J. Clin. Invest. 2004, 114, 468−471. (5) Kalos, M.; Levine, B. L.; Porter, D. L.; Katz, S.; Grupp, S. A.; Bagg, A.; June, C. H. Sci. Transl. Med. 2011, 3, 95ra73. (6) Porter, D. L.; Levine, B. L.; Kalos, M.; Bagg, A.; June, C. H. N. Engl. J. Med. 2011, 365, 725−733. (7) 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. Sci. Transl. Med. 2015, 7, 303ra139. (8) Butler, M. O.; Hirano, N. Immunol. Rev. 2014, 257, 191−209. (9) Crompton, J. G.; Sukumar, M.; Restifo, N. P. Immunol. Rev. 2014, 257, 264−276. (10) Milone, M. C.; Kam, L. C. WIREs Nanomed. Nanobiotechnol. 2013, 5, 75−85. (11) Platzman, I.; Janiesch, J.-W.; Matic, J.; Spatz, J. P. Isr. J. Chem. 2013, 53, 655−669. (12) Paul, W. E.; Seder, R. A. Cell 1994, 76, 241−251. (13) Yamada, S.; Nelson, W. J. Annu. Rev. Biochem. 2007, 76, 267− 294. (14) Grakoui, A.; Bromley, S. K.; Sumen, C.; Davis, M. M.; Shaw, A. S.; Allen, P. M.; Dustin, M. L. Science 1999, 285, 221−226. (15) Linsley, P. S.; Brady, W.; Grosmaire, L.; Aruffo, A.; Damle, N. K.; Ledbetter, J. A. J. Exp. Med. 1991, 173, 721−730. (16) Koulova, L.; Clark, E. A.; Shu, G.; Dupont, B. J. Exp. Med. 1991, 173, 759−762. (17) Linsley, P. S.; Clark, E. A.; Ledbetter, J. A. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 5031−5035. (18) June, C. H.; Ledbetter, J. A.; Linsley, P. S.; Thompson, C. B. Immunol. Today 1990, 11, 211−216. (19) Wuelfing, C.; Davis, M. M. Science 1998, 282, 2266−2269. (20) Dustin, M. L.; Bromley, S. K.; Kan, Z.; Peterson, D. A.; Unanue, E. R. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 3909−3913. (21) Tsoukas, C. D.; Landgraf, B.; Bentin, J.; Valentine, M.; Lotz, M.; Vaughan, J. H.; Carson, D. A. J. Immunol. 1985, 135, 1719−1723. (22) Stingl, L. A.; Sinska, A.; Landesmann, U.; Smolen, J. S. Clin. Exp. Immunol. 1987, 68, 146−155. (23) June, C. H.; Ledbetter, J. A.; Gillespie, M. M.; Lindsten, T.; Thompson, C. B. Mol. Cell. Biol. 1987, 7, 4472−4481. (24) Dustin, M. L.; Springer, T. A. Nature 1989, 341, 619−624. (25) Masopust, D.; Schenkel, J. M. Nat. Rev. Immunol. 2013, 13, 309−320. (26) Hamann, A.; Engelhardt, B. Leukocyte Trafficking: Molecular Mechanisms, Therapeutic Targets, and Methods; Wiley-VCH Verlag GmbH & Co.: Weinheim, Germany, 2005. (27) Bradley, L. M.; Watson, S. R. Curr. Opin. Immunol. 1996, 8, 312−320. (28) Brezinschek, R. I.; Lipsky, P. E.; Galea, P.; R, R. V.; Oppenheimer-Marks, N. J. Immunol. 1995, 154, 3062−3077. (29) Brown, F. D.; Turley, S. J. J. Immunol. 2015, 194, 1389−1394. (30) Springer, T. A. Nature 1990, 346, 425−434. (31) Davis, L. S.; Oppenheimer-Marks, N.; Bednarczyk, J. L.; McIntyre, B. W.; Lipsky, P. E. J. Immunol. 1990, 145, 785−793. (32) 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. Biochim. Biophys. Acta, Mol. Cell Res. 2007, 1773, 176−184. (33) Humphries, J. D.; Byron, A.; Humphries, M. J. J. Cell Sci. 2006, 119, 3901−3903. G
DOI: 10.1021/acs.nanolett.7b02636 Nano Lett. XXXX, XXX, XXX−XXX
