Research Article For reprint orders, please contact: [email protected]
Nanoparticles and antigen-specific T-cell therapeutics: a comprehensive study on uptake and release
Aim: T lymphocytes are used as cellular therapeutics in many disease entities including cancer. We investigated the uptake and retention of nanoparticles (NPs) by these nonphagocytic cells. Materials & methods: Uptake, release and toxicity of various polymeric NPpreparations were analyzed by flow cytometry, confocal laser scanning microscopy and transmission electron microscopy. T-cell effector functions were measured using IFN-γ-ELISPOT and 51Chromium-release assays. Results: Aminofunctionalized NPs were efficiently ingested by antigen-specific T cells without adversely influencing effector functions. NPs were stored in membrane-surrounded vesicles, with major proportions released extracellularly during 24 h. Conclusion: Amino-functionalized polymeric NPs are efficiently taken up by human T cells and could be used to design nanocarriers for direct access and manipulation of antigen-specific T cells in vivo. Keywords: cell imaging • cellular therapy • drug delivery • endocytosis • leukemia • nanoparticles • NP release • T lymphocytes • tumor
Cell-mediated immune responses against bacteria and viruses are mainly exerted by T lymphocytes. T lymphocytes also include very effective cell populations for eliciting and controlling immune reactions against tumors and leukemias [1,2] . During autoimmune diseases as well as allo-transplant rejection, T lymphocytes can either induce damage to organs or mediate tolerance by downregulating an immune cell attack [3,4] . For therapeutic approaches ex vivo manipulated or expanded T-cell therapeutics find increasing use. There are several subtypes of T lymphocytes of which the two most important ones will be addressed herein: CD4 + T helper cells assist other leukocytes in immune cell reactions, including the development of B cells into plasma cells and memory B cells as well as the activation of cytotoxic T lymphocytes (CTLs) and macrophages [5] . CD8 + T cells represent another major cell type of specific immunity. They have a crucial role in the clearance of virally infected cells and tumor cells, and in the rejection of allogeneic cells
10.2217/NNM.14.160 © 2015 Future Medicine Ltd
from organ transplants [6] . With T lymphocytes taking this central position in the immunological network it is easily comprehensible that manipulating them is an important part in nearly every immunotherapeutic strategy, aiming either to strengthen immune reactions (e.g., in infection and tumor) or to reduce excessive immune responses (e.g., in transplant rejection and autoimmunity). Genetic reprogramming is a promising strategy for manipulation of T cells. The approach is based on the introduction of genetic material into T cells, allowing the modulation of trafficking, homing, persistence and effector functions of these cells in vivo. Efficient genetic manipulation of T cells can be achieved by several transfection methods in vitro. However, none of them can be used directly in patients in vivo. Here nanoparticles (NPs) could be cutting edge tools, because they cannot only transport contrast agents, drugs and nucleic acids, but can target a cell type of interest in vivo by employing surface functionalization [7,8] .
Nanomedicine (Lond.) (2015) 10(7), 1063–1076
Oliver Zupke1,2, Eva Distler1, Anna Jürchott1, Umaporn Paiphansiri3, Martin Dass3, Simone Thomas1,4, Udo F Hartwig1, Matthias Theobald1, Katharina Landfester3, Volker Mailänder*,‡,1,3 & Wolfgang Herr**,‡,1,4, 1 Department of Medicine III, Hematology, Oncology & Pneumology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany 2 Fraunhofer ICT-IMM, Carl-Zeiss-Str 18–20, 55129 Mainz, Germany 3 Max Planck Institute for Polymer Research, Ackermannweg 10, 55129 Mainz, Germany 4 Department of Medicine III, Hematology & Internal Oncology, University Hospital of Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany *Author for correspondence: Tel.: +49 6131 17 6299 Fax: +49 6131 17 5546 volker.mailaender@ unimedizin-mainz.de **Author for correspondence: Tel.: +49 941 944 5501 Fax: +49 941 944 5502 wolfgang.herr@ ukr.de ‡ Authors contributed equally
part of
ISSN 1743-5889
1063
Research Article Zupke, Distler, Jürchott et al. Polymeric NP materials with biodegradability even provide the opportunity to customize drug delivery and release. Hereby, time point, duration and intracellular localization for release can be designed as needed during the synthesis of the NPs [9] . Most literature deals with malignant or transformed cell lines and how NPs are taken up by these cells [10–12] . Primary T cells have been rarely used and for NP uptake only few publications are available. This work shows that iron oxide-loaded T cells can be visualized by MRI upon adoptive transfer into live animals and can be detected at sites of tumor infiltration or autoimmune disease [13–15] . As these papers focused on the proof of concept and detectability of T cells, it was only rarely addressed whether NP loading influences the function of T cells [16,17] . While most investigators showed that particle labeling persists over several days [14,18] , Smirnov et al. reported an approximately 40% loss of MRI signals during the first 24 h due to NP dilution in dividing T cells [19] . Also, employing T cells as transport vehicles to deliver chemotherapeutic drugs to tumor sites has been proposed [20] . As this work has been done in rodents it remains unclear whether it can be reproduced with human T cells. Even with the use of transfection agents, uptake of NPs in human T cells is considerably less efficient than in other cell types [17,21] . We therefore analyzed newly designed fluorescent polymeric NPs and identified amino-functionalized ones that are incorporated into primary human CD4 + and CD8 + T cells in nearly the same order of magnitude as we have recently shown for professional antigen-presenting cells (APC) such as dendritic cells (DCs) [22] . Comprehensive in vitro studies defined experimental conditions in which T-cell effector functions are fully preserved upon NP loading. Interestingly, particle uptake was strongly inhibited if human serum (HS) was present. We also show that human T cells released most NPs during the first 24 h.
irradiated (100 Gy) Epstein-Barr Virus (EBV) transformed B-lymphoblastoid cell lines (B-LCL) in AIM-V medium (Gibco/Invitrogen, NY, USA) supplemented with 10% HS. Recombinant human rh IL-2 (Novartis, Switzerland) at 100 U/ml was added on day (d) 3. T cells were restimulated weekly in fresh cytokine-containing medium. Leukemia-reactive CTL clones were generated from naive CD8 + T cells of healthy donors by in vitro stimulation with HLA-identical acute myeloid leukemia (AML) blasts as described [24,25] . Renal-cell carcinoma (RCC)-reactive CD8 + CTLs were generated by in vitro stimulation of healthy donor CD8 + T cells with allogeneic HLA-identical RCC cells [26] . Splenocytes derived from C57BL/6 mice were used to isolate murine CD4 + and CD8 + T cells by immunomagnetic cell separation (Miltenyi Biotec, Germany). Purified (>95%) T cells were polyclonally stimulated with immobilized anti-CD3 mAb (Cedarlane, Canada) at 0.8–1 × 107 cells/well in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum (FCS) and 100 U/ml rhIL-2 for 72 h.
Materials & methods
Analysis of NP uptake & toxicity by flow cytometric analysis
NP synthesis
Composite latex particles with incorporated fluorescent dye were produced by miniemulsion polymerization [23] . For details on synthesis and characterization see the Supplementary Material (available online at: www.futuremedicine.com/doi/full/10.2217/ NNM.14.160). T-cell cultures
Alloreactive primary human CD4 + and CD8 + T cells were generated by stimulation of immunomagnetically (Miltenyi Biotec, Germany) isolated CD4 + or CD8 + T cells from peripheral blood mononuclear cells (PBMC) of healthy donors with HLA-disparate
1064
Nanomedicine (Lond.) (2015) 10(7)
Loading of T cells with NPs
For labeling with NPs, 3 × 105 T cells were seeded in 1 ml AIM-V medium supplemented with 1% HS in 48-well plates at day 2 after last antigen-specific restimulation. NPs were added at a concentration of 75 μg/ml and incubated with T cells for 16 h at 37°C. Afterward, T cells were spun down to remove excess particles in supernatant and NP-loaded cells were resuspended in 300 μl fresh medium for subsequent use. In experiments determining the concentration dependence of NP uptake, particles were added at concentrations from 25–2400 μg/ml. To identify the optimal incubation time, T cells were loaded with particles for periods from 2–24 h. Murine T cells were incubated with NPs in RPMI medium supplemented with 5% FCS and 100 U/ml rhIL-2 at day 3 of stimulation.
Fluorescence-activated cell sorting (FACS) measurements were performed on BD FACSCantoTM flow cytometer equipped with FACSDivaTM software (BD Biosciences, CA, USA). Fluorescence intensity of N-(2,6-diisopropylphenyl)perylene-3,4 dicarbonacidimide (PMI) containing particles taken up by alloreactive T cells and clones was measured in FL1 channel using 530/30 nm filter. Staining with 12.5 μg/ml 7-aminoactinomycin D (7-AAD, Sigma-Aldrich, Germany) for 15 min was used to identify dying and dead cells. For analyzing particle uptake, only viable T cells (7-AADneg /lymphocyte gate) were gated. The median fluorescence intensity (MFI) of the FL1 channel was used to
future science group
Nanoparticle labeling of human T lymphocytes
determine particle uptake. Results represent means of duplicates with standard deviations (SDs) indicated. Data were analyzed for statistical significance by onetailed paired Student’s t-test (*p < 0.05 and **p < 0.01 vs respective controls). As particles contained different amounts of fluorescent dye (incorporated dye [icDye] given as w/w), the FL1-MFI was normalized by the following formula in order to compare the efficiency of uptake between different particle types: Normalized FL1 = nFL1(particle x) = Median FL1(particle x) Median FL1(uncharged particle)
icDye(particle x) icDye(uncharged particle)
This normalized value is reported as relative fluorescence intensity as seen in Supplementary Figure 1. Confocal laser scanning microscopy
Intracellular localization of particles was confirmed with Zeiss laser scanning microscopy (LSM) 710 NLO confocal LSM (cLSM) equipped with Plan-Apochromat 63×/1.4 Oil DIC objective and Zen 2009 Software (Carl Zeiss, Germany). After removal of nonincorporated particles by centrifugation, nuclear staining was performed by incubating cells with 200 nM Hoechst 33342TM (Invitrogen) for 30 min at 37°C. Immediately before microscopy, cell membranes were stained with 1.67 μg/ml CellMaskTM Orange plasma membrane stain (Invitrogen). For colocalization studies, T cells were intracellularly stained (Cytofix/Cytoperm Protocol, BD Biosciences) with mAbs for endosomal proteins EEA1, Lamp1, Rab5, Rab7, flotillin-2 and Rab11 (BD Biosciences and Abcam, MA, USA), respectively, followed by fluorochrome-conjugated secondary antibodies (Molecular Probes, OR, USA and Invitrogen). CellMaskTM Orange and Alexa FluorTM 555 and 546 dyes (Life Technologies, CA, USA) were excited by 543 nm helium-neon laser. The argon laser (488 nm) was used for excitation of PMI, and Hoechst 33342TM was excited by the blue violet diode laser (405 nm). Samples for transmission electron microscopy (TEM) were prepared as described in Supplementary Material. T-cell assays
Standard 5-h 51Chromium (51Cr)-release assays and 20 h IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays were performed in duplicate wells as described [24] . Pharmacological inhibition of endocytotic pathways
For analyzing the NP-uptake mechanism, T cells were incubated with NPs in the presence of pharma-
future science group
Research Article
cological drugs that block components of the three major endocytotic pathways: macropinocytosis, clathrin- and caveolin-mediated endocytosis. In dosefinding studies, T cells were incubated with different concentrations of dynasore (1–160 μM), chlorpromazine (6.13–100 μM), 5-(N-ethyl-N-isopropyl) amiloride (EIPA; 1.25–160 mM) and cytochalasin D (0.25–20 μM) to determine the maximum nontoxic concentration that could be used for inhibition studies. Cell viability was analyzed using 7-AAD staining. In subsequent inhibition experiments, CD4 + and CD8 + T cells were preincubated with drugs in phosphate-buffered saline (PBS) containing calcium chloride and magnesium chloride for 30 min at 37°C. Subsequently, NPs (75 μg/ml) were added and incubated with T cells in presence of inhibitors for further 60 min at 37°C. NP incubation time was reduced to 60 min in these experiments, because blocking of one endocytotic pathway could allow for NP incorporation via other routes. Pretreatment of NPs with inactivated influenza A whole virus particles
Amino-functionalized NPs were preincubated with a 244.5 μg/ml (hemagglutinin protein content) preparation of an inactivated influenza A (H3N2) wholevirus vaccine (Baxter, Austria) for 0.5 h at 37°C allowing an electrostatic attachment of whole virus on NP surfaces (1.91 μg whole virus per 75 μg/ml NPs). Afterward, the NP–virus complexes were incubated with DCs or CD4 + T lymphocytes in serumfree medium for further 2 h at 37°C. After removal of excess particles by centrifugation, NP-loaded cells were recultured in particle-free medium for follow-up over 9 days. NP uptake and release were analyzed by flow cytometry. Statistical analysis
Student’s t-test was used for demonstrating significance levels. Significance levels are given as * for p 95% for at least 24 h (not shown). The results are representative of at least two independent experiments with cells from one donor. Data are presented as described in Figure 1. Conc.: Concentration; HS: Human serum; NC: Negative control (without nanoparticles); NP: Nanoparticle.
future science group
www.futuremedicine.com
1071
Research Article Zupke, Distler, Jürchott et al.
16,000 14,000 12,000 10,000 8000 6000 4000 2000 0
** d1 d2 d3 NC
NP
Median fluorescence intensity
C
NP + virus
8000 6000 4000
d1 d2 d3
2000 0
NC
* ** ** ** *
60,000 50,000
***
40,000 30,000
d1 d2 d3 d6 d9
n.s.
20,000 10,000 0
NC
NP
NP + virus
D
**
10,000
Median fluorescence intensity
B * * * **
EBV-B LCL + NP
Median fluorescence intensity
Median fluorescence intensity
A
10,000
**
8000 **
6000 4000
d1 d2 d3
2000 0
NC
CD4 (MUR) CD8 (MUR) + NP + NP
Figure 6. Improved delivery of nanoparticles into T lymphocytes and dendritic cells after ‘biocoating’ with whole virus particles and uptake of nanoparticles by antigen-presenting B lymphocytes as well as murine T cells. For viral biocoating, NP-1-NH2 (75 μg/ml) were pretreated with an influenza inactivated whole virus vaccine preparation for 0.5 h at 37°C and were then incubated with immune cells for further 2 h at 37°C. Residual NP- loaded cells were recultured in particle-free medium for monitoring NP content at indicated time points. Data in (A & B) are derived from CD4 + T cells and dendritic cells, respectively. (C) To investigate if NP release may be attributable either to the lymphocytic origin of T cells or to the missing antigen-presenting function, B-lymphoblastoid cells that combine both features were loaded with 75 μg/ml NP-1-NH2 for 16 h, then washed and followed for NP content. (D) To exclude that nanoparticle release is a species-specific property of human T lymphocytes, murine CD4 + and CD8 + T cells were also analyzed for NP uptake and release after labeling with 75 μg/ml NP-1-NH2 for 16 h. The results are representative of at least two independent experiments each. Data are presented as described in Figure 1. d: Day(s); MUR: Murine; NC: Negative control (without nanoparticles); NP: Nanoparticle; ns: Not significant.
culture periods. This would also be advantageous for T-cell therapeutics itself, as long-term in vitro culture of tumor- and virus-reactive T lymphocytes have been associated with diminished effector function, persistence and homing upon adoptive transfer in vivo [39] . In summary, expansion of antigen-specific T cells over few weeks in vitro seems to be optimal with regard to nanoparticle uptake as well as for maintaining key functions and in vivo performance of labeled cells after infusion into patients. Consistent with other cell types [12,40,41] , T cells showed reduced NP ingestion at 4°C when compared with 37°C. We thus assumed that an energydependent uptake process with endocytotic pathways might be involved. Next we used pharmacologic endocytosis inhibitors to identify potential mechanisms of NP uptake in T cells. We had successfully implemented this approach in HeLa cells previously [12] . With this we observed some inhibitory effect (i.e.,
Nanoparticles and antigen-specific T-cell therapeutics: a comprehensive study on uptake and release
Aim: T lymphocytes are used as cellular therapeutics in many disease entities including cancer. We investigated the uptake and retention of nanoparticles (NPs) by these nonphagocytic cells. Materials & methods: Uptake, release and toxicity of various polymeric NPpreparations were analyzed by flow cytometry, confocal laser scanning microscopy and transmission electron microscopy. T-cell effector functions were measured using IFN-γ-ELISPOT and 51Chromium-release assays. Results: Aminofunctionalized NPs were efficiently ingested by antigen-specific T cells without adversely influencing effector functions. NPs were stored in membrane-surrounded vesicles, with major proportions released extracellularly during 24 h. Conclusion: Amino-functionalized polymeric NPs are efficiently taken up by human T cells and could be used to design nanocarriers for direct access and manipulation of antigen-specific T cells in vivo. Keywords: cell imaging • cellular therapy • drug delivery • endocytosis • leukemia • nanoparticles • NP release • T lymphocytes • tumor
Cell-mediated immune responses against bacteria and viruses are mainly exerted by T lymphocytes. T lymphocytes also include very effective cell populations for eliciting and controlling immune reactions against tumors and leukemias [1,2] . During autoimmune diseases as well as allo-transplant rejection, T lymphocytes can either induce damage to organs or mediate tolerance by downregulating an immune cell attack [3,4] . For therapeutic approaches ex vivo manipulated or expanded T-cell therapeutics find increasing use. There are several subtypes of T lymphocytes of which the two most important ones will be addressed herein: CD4 + T helper cells assist other leukocytes in immune cell reactions, including the development of B cells into plasma cells and memory B cells as well as the activation of cytotoxic T lymphocytes (CTLs) and macrophages [5] . CD8 + T cells represent another major cell type of specific immunity. They have a crucial role in the clearance of virally infected cells and tumor cells, and in the rejection of allogeneic cells
10.2217/NNM.14.160 © 2015 Future Medicine Ltd
from organ transplants [6] . With T lymphocytes taking this central position in the immunological network it is easily comprehensible that manipulating them is an important part in nearly every immunotherapeutic strategy, aiming either to strengthen immune reactions (e.g., in infection and tumor) or to reduce excessive immune responses (e.g., in transplant rejection and autoimmunity). Genetic reprogramming is a promising strategy for manipulation of T cells. The approach is based on the introduction of genetic material into T cells, allowing the modulation of trafficking, homing, persistence and effector functions of these cells in vivo. Efficient genetic manipulation of T cells can be achieved by several transfection methods in vitro. However, none of them can be used directly in patients in vivo. Here nanoparticles (NPs) could be cutting edge tools, because they cannot only transport contrast agents, drugs and nucleic acids, but can target a cell type of interest in vivo by employing surface functionalization [7,8] .
Nanomedicine (Lond.) (2015) 10(7), 1063–1076
Oliver Zupke1,2, Eva Distler1, Anna Jürchott1, Umaporn Paiphansiri3, Martin Dass3, Simone Thomas1,4, Udo F Hartwig1, Matthias Theobald1, Katharina Landfester3, Volker Mailänder*,‡,1,3 & Wolfgang Herr**,‡,1,4, 1 Department of Medicine III, Hematology, Oncology & Pneumology, University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstr. 1, 55131 Mainz, Germany 2 Fraunhofer ICT-IMM, Carl-Zeiss-Str 18–20, 55129 Mainz, Germany 3 Max Planck Institute for Polymer Research, Ackermannweg 10, 55129 Mainz, Germany 4 Department of Medicine III, Hematology & Internal Oncology, University Hospital of Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany *Author for correspondence: Tel.: +49 6131 17 6299 Fax: +49 6131 17 5546 volker.mailaender@ unimedizin-mainz.de **Author for correspondence: Tel.: +49 941 944 5501 Fax: +49 941 944 5502 wolfgang.herr@ ukr.de ‡ Authors contributed equally
part of
ISSN 1743-5889
1063
Research Article Zupke, Distler, Jürchott et al. Polymeric NP materials with biodegradability even provide the opportunity to customize drug delivery and release. Hereby, time point, duration and intracellular localization for release can be designed as needed during the synthesis of the NPs [9] . Most literature deals with malignant or transformed cell lines and how NPs are taken up by these cells [10–12] . Primary T cells have been rarely used and for NP uptake only few publications are available. This work shows that iron oxide-loaded T cells can be visualized by MRI upon adoptive transfer into live animals and can be detected at sites of tumor infiltration or autoimmune disease [13–15] . As these papers focused on the proof of concept and detectability of T cells, it was only rarely addressed whether NP loading influences the function of T cells [16,17] . While most investigators showed that particle labeling persists over several days [14,18] , Smirnov et al. reported an approximately 40% loss of MRI signals during the first 24 h due to NP dilution in dividing T cells [19] . Also, employing T cells as transport vehicles to deliver chemotherapeutic drugs to tumor sites has been proposed [20] . As this work has been done in rodents it remains unclear whether it can be reproduced with human T cells. Even with the use of transfection agents, uptake of NPs in human T cells is considerably less efficient than in other cell types [17,21] . We therefore analyzed newly designed fluorescent polymeric NPs and identified amino-functionalized ones that are incorporated into primary human CD4 + and CD8 + T cells in nearly the same order of magnitude as we have recently shown for professional antigen-presenting cells (APC) such as dendritic cells (DCs) [22] . Comprehensive in vitro studies defined experimental conditions in which T-cell effector functions are fully preserved upon NP loading. Interestingly, particle uptake was strongly inhibited if human serum (HS) was present. We also show that human T cells released most NPs during the first 24 h.
irradiated (100 Gy) Epstein-Barr Virus (EBV) transformed B-lymphoblastoid cell lines (B-LCL) in AIM-V medium (Gibco/Invitrogen, NY, USA) supplemented with 10% HS. Recombinant human rh IL-2 (Novartis, Switzerland) at 100 U/ml was added on day (d) 3. T cells were restimulated weekly in fresh cytokine-containing medium. Leukemia-reactive CTL clones were generated from naive CD8 + T cells of healthy donors by in vitro stimulation with HLA-identical acute myeloid leukemia (AML) blasts as described [24,25] . Renal-cell carcinoma (RCC)-reactive CD8 + CTLs were generated by in vitro stimulation of healthy donor CD8 + T cells with allogeneic HLA-identical RCC cells [26] . Splenocytes derived from C57BL/6 mice were used to isolate murine CD4 + and CD8 + T cells by immunomagnetic cell separation (Miltenyi Biotec, Germany). Purified (>95%) T cells were polyclonally stimulated with immobilized anti-CD3 mAb (Cedarlane, Canada) at 0.8–1 × 107 cells/well in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum (FCS) and 100 U/ml rhIL-2 for 72 h.
Materials & methods
Analysis of NP uptake & toxicity by flow cytometric analysis
NP synthesis
Composite latex particles with incorporated fluorescent dye were produced by miniemulsion polymerization [23] . For details on synthesis and characterization see the Supplementary Material (available online at: www.futuremedicine.com/doi/full/10.2217/ NNM.14.160). T-cell cultures
Alloreactive primary human CD4 + and CD8 + T cells were generated by stimulation of immunomagnetically (Miltenyi Biotec, Germany) isolated CD4 + or CD8 + T cells from peripheral blood mononuclear cells (PBMC) of healthy donors with HLA-disparate
1064
Nanomedicine (Lond.) (2015) 10(7)
Loading of T cells with NPs
For labeling with NPs, 3 × 105 T cells were seeded in 1 ml AIM-V medium supplemented with 1% HS in 48-well plates at day 2 after last antigen-specific restimulation. NPs were added at a concentration of 75 μg/ml and incubated with T cells for 16 h at 37°C. Afterward, T cells were spun down to remove excess particles in supernatant and NP-loaded cells were resuspended in 300 μl fresh medium for subsequent use. In experiments determining the concentration dependence of NP uptake, particles were added at concentrations from 25–2400 μg/ml. To identify the optimal incubation time, T cells were loaded with particles for periods from 2–24 h. Murine T cells were incubated with NPs in RPMI medium supplemented with 5% FCS and 100 U/ml rhIL-2 at day 3 of stimulation.
Fluorescence-activated cell sorting (FACS) measurements were performed on BD FACSCantoTM flow cytometer equipped with FACSDivaTM software (BD Biosciences, CA, USA). Fluorescence intensity of N-(2,6-diisopropylphenyl)perylene-3,4 dicarbonacidimide (PMI) containing particles taken up by alloreactive T cells and clones was measured in FL1 channel using 530/30 nm filter. Staining with 12.5 μg/ml 7-aminoactinomycin D (7-AAD, Sigma-Aldrich, Germany) for 15 min was used to identify dying and dead cells. For analyzing particle uptake, only viable T cells (7-AADneg /lymphocyte gate) were gated. The median fluorescence intensity (MFI) of the FL1 channel was used to
future science group
Nanoparticle labeling of human T lymphocytes
determine particle uptake. Results represent means of duplicates with standard deviations (SDs) indicated. Data were analyzed for statistical significance by onetailed paired Student’s t-test (*p < 0.05 and **p < 0.01 vs respective controls). As particles contained different amounts of fluorescent dye (incorporated dye [icDye] given as w/w), the FL1-MFI was normalized by the following formula in order to compare the efficiency of uptake between different particle types: Normalized FL1 = nFL1(particle x) = Median FL1(particle x) Median FL1(uncharged particle)
icDye(particle x) icDye(uncharged particle)
This normalized value is reported as relative fluorescence intensity as seen in Supplementary Figure 1. Confocal laser scanning microscopy
Intracellular localization of particles was confirmed with Zeiss laser scanning microscopy (LSM) 710 NLO confocal LSM (cLSM) equipped with Plan-Apochromat 63×/1.4 Oil DIC objective and Zen 2009 Software (Carl Zeiss, Germany). After removal of nonincorporated particles by centrifugation, nuclear staining was performed by incubating cells with 200 nM Hoechst 33342TM (Invitrogen) for 30 min at 37°C. Immediately before microscopy, cell membranes were stained with 1.67 μg/ml CellMaskTM Orange plasma membrane stain (Invitrogen). For colocalization studies, T cells were intracellularly stained (Cytofix/Cytoperm Protocol, BD Biosciences) with mAbs for endosomal proteins EEA1, Lamp1, Rab5, Rab7, flotillin-2 and Rab11 (BD Biosciences and Abcam, MA, USA), respectively, followed by fluorochrome-conjugated secondary antibodies (Molecular Probes, OR, USA and Invitrogen). CellMaskTM Orange and Alexa FluorTM 555 and 546 dyes (Life Technologies, CA, USA) were excited by 543 nm helium-neon laser. The argon laser (488 nm) was used for excitation of PMI, and Hoechst 33342TM was excited by the blue violet diode laser (405 nm). Samples for transmission electron microscopy (TEM) were prepared as described in Supplementary Material. T-cell assays
Standard 5-h 51Chromium (51Cr)-release assays and 20 h IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays were performed in duplicate wells as described [24] . Pharmacological inhibition of endocytotic pathways
For analyzing the NP-uptake mechanism, T cells were incubated with NPs in the presence of pharma-
future science group
Research Article
cological drugs that block components of the three major endocytotic pathways: macropinocytosis, clathrin- and caveolin-mediated endocytosis. In dosefinding studies, T cells were incubated with different concentrations of dynasore (1–160 μM), chlorpromazine (6.13–100 μM), 5-(N-ethyl-N-isopropyl) amiloride (EIPA; 1.25–160 mM) and cytochalasin D (0.25–20 μM) to determine the maximum nontoxic concentration that could be used for inhibition studies. Cell viability was analyzed using 7-AAD staining. In subsequent inhibition experiments, CD4 + and CD8 + T cells were preincubated with drugs in phosphate-buffered saline (PBS) containing calcium chloride and magnesium chloride for 30 min at 37°C. Subsequently, NPs (75 μg/ml) were added and incubated with T cells in presence of inhibitors for further 60 min at 37°C. NP incubation time was reduced to 60 min in these experiments, because blocking of one endocytotic pathway could allow for NP incorporation via other routes. Pretreatment of NPs with inactivated influenza A whole virus particles
Amino-functionalized NPs were preincubated with a 244.5 μg/ml (hemagglutinin protein content) preparation of an inactivated influenza A (H3N2) wholevirus vaccine (Baxter, Austria) for 0.5 h at 37°C allowing an electrostatic attachment of whole virus on NP surfaces (1.91 μg whole virus per 75 μg/ml NPs). Afterward, the NP–virus complexes were incubated with DCs or CD4 + T lymphocytes in serumfree medium for further 2 h at 37°C. After removal of excess particles by centrifugation, NP-loaded cells were recultured in particle-free medium for follow-up over 9 days. NP uptake and release were analyzed by flow cytometry. Statistical analysis
Student’s t-test was used for demonstrating significance levels. Significance levels are given as * for p 95% for at least 24 h (not shown). The results are representative of at least two independent experiments with cells from one donor. Data are presented as described in Figure 1. Conc.: Concentration; HS: Human serum; NC: Negative control (without nanoparticles); NP: Nanoparticle.
future science group
www.futuremedicine.com
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Research Article Zupke, Distler, Jürchott et al.
16,000 14,000 12,000 10,000 8000 6000 4000 2000 0
** d1 d2 d3 NC
NP
Median fluorescence intensity
C
NP + virus
8000 6000 4000
d1 d2 d3
2000 0
NC
* ** ** ** *
60,000 50,000
***
40,000 30,000
d1 d2 d3 d6 d9
n.s.
20,000 10,000 0
NC
NP
NP + virus
D
**
10,000
Median fluorescence intensity
B * * * **
EBV-B LCL + NP
Median fluorescence intensity
Median fluorescence intensity
A
10,000
**
8000 **
6000 4000
d1 d2 d3
2000 0
NC
CD4 (MUR) CD8 (MUR) + NP + NP
Figure 6. Improved delivery of nanoparticles into T lymphocytes and dendritic cells after ‘biocoating’ with whole virus particles and uptake of nanoparticles by antigen-presenting B lymphocytes as well as murine T cells. For viral biocoating, NP-1-NH2 (75 μg/ml) were pretreated with an influenza inactivated whole virus vaccine preparation for 0.5 h at 37°C and were then incubated with immune cells for further 2 h at 37°C. Residual NP- loaded cells were recultured in particle-free medium for monitoring NP content at indicated time points. Data in (A & B) are derived from CD4 + T cells and dendritic cells, respectively. (C) To investigate if NP release may be attributable either to the lymphocytic origin of T cells or to the missing antigen-presenting function, B-lymphoblastoid cells that combine both features were loaded with 75 μg/ml NP-1-NH2 for 16 h, then washed and followed for NP content. (D) To exclude that nanoparticle release is a species-specific property of human T lymphocytes, murine CD4 + and CD8 + T cells were also analyzed for NP uptake and release after labeling with 75 μg/ml NP-1-NH2 for 16 h. The results are representative of at least two independent experiments each. Data are presented as described in Figure 1. d: Day(s); MUR: Murine; NC: Negative control (without nanoparticles); NP: Nanoparticle; ns: Not significant.
culture periods. This would also be advantageous for T-cell therapeutics itself, as long-term in vitro culture of tumor- and virus-reactive T lymphocytes have been associated with diminished effector function, persistence and homing upon adoptive transfer in vivo [39] . In summary, expansion of antigen-specific T cells over few weeks in vitro seems to be optimal with regard to nanoparticle uptake as well as for maintaining key functions and in vivo performance of labeled cells after infusion into patients. Consistent with other cell types [12,40,41] , T cells showed reduced NP ingestion at 4°C when compared with 37°C. We thus assumed that an energydependent uptake process with endocytotic pathways might be involved. Next we used pharmacologic endocytosis inhibitors to identify potential mechanisms of NP uptake in T cells. We had successfully implemented this approach in HeLa cells previously [12] . With this we observed some inhibitory effect (i.e.,
