Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy.

The amount and density of MHC-Ig and anti-CD28 on the surface of micro-(cell-sized) and nano-aAPC. Protein was quantified as described in Supplementary Figure 1, and particle concentration determined by counts (micro-aAPC) or Nanoparticle Tracking Analysis (nano-aAPC). High (HD) and low (LD) density particles were synthesized by varying amount of protein per particle during synthesis. Signal 1 nanoparticles were synthesized without anti-CD28.

Supplementary Figure 2. pMEL T cell Proliferation Induced by Micro-aAPC
(A) CD8+ pMEL splenocytes include a population of memory-phenotype, CD44 positive cells (representative percentage shown as percentage of CD8, left). CD44lo naive cells were isolated by a notouch negative selection enrichment with anti-CD44 antibody in a magnetic enrichment column. (B) Proliferation of Naive CD44lo (left) and activated (right) cells by CFSE dilution stimulated three days with micro-aAPC (dark red and blue lines) and nano-aAPC (light red and blue lines) or unstimulated (grey lines). Micro-and nano-aAPC were used at doses presenting equivalent total amount of MHC-Ig (8 ng). Nano-aAPC data are re-produced from The mean of this distribution is ‫ܧ‬ሾ‫ݐ‬ሿ ൌ ݊ ‫ݎ‬ ⁄ . If MHC-Ig dimer is assumed to make one contact with a naive T cell 1 , then r can be estimated from the off-rate of MHC-Ig on naive cells (8.9×10 -3 ). Thus, for any given condition, ‫ܧ‬ሾ‫ݐ‬ሿ is derived from the half-life of MHC-Ig dimer or particle on naive or active cells (‫ݐ‬ଵ ଶ ൗ ሻ, and r is assumed constant. The number of TCR-MHC contacts is estimated as n: The true number of contacts is likely to be higher than this estimate, as MHC-Ig are likely to make more than one contact with naive cells.

Preparation of MHC-Ig Dimers
Soluble MHC-Ig dimers, K b -Ig and D b -Ig, were prepared and loaded with peptide as described 2 . Briefly, K b -Ig molecules were loaded with peptide by stripping at alkaline condition (pH 11.5), and then refolded in the presence of 50 fold molar excess peptide. D b -Ig molecules were stripped under mildly acidic conditions (pH 6.5) and refolded in the presence of 50 fold molar excess peptide and 2-fold molar excess of human β 2 -microglobulin 3 . Peptides SIY (SIYRYYGL, synthetic), SIIN (SIINFEKL, derived from ovalbumin protein), GP100 (KVPRNQDWL, from melanocyte GP100 protein) and ASN (ASNENMETH, from influenza A nucleoprotein) were purchased from Genscript (Piscataway, NJ). Protein concentration was determined after labeling by size exclusion high performance liquid chromatography (HPLC).

Micro-aAPC Synthesis
Micro-aAPCs were fabricated as described previously 4

Nanoparticle Tracking Analysis
A Nanosight LM10 equipped with a sensitive CCD camera was used for characterizing the size distribution of nano-aAPC by NTA. 50 µL of diluted nanoparticle solution was loaded into the sample chamber, which was connected to a 405 nm laser source. A 60 s movie containing the Brownian motion tracking of the scattering centroids (particles) was recorded using NTA software (Version 2.0). The movie was processed using the manufacturer recommended auto settings with manual adjustment of the gain, blur and brightness as recommended. The nanoparticle solution was diluted in phosphate buffered saline to adjust the sample concentration to 5×10 12 particles mL −1 .

Micro-aAPC Microscopy
T cells were incubated with micro-aAPC, spun at 1000 RPM for 1 minute to pack cells and particles, and incubated for 60 minutes at 4 o C. Cells were subsequently transferred to a 96-well plate at 37 o C in the presence or absence of a magnetic field generated by Neodymium N52 disk magnets. After 30 minutes, cells were washed and stained at 4 o C with Alexa488 anti-LFA1, monoclonal PE anti-mouse IgG1, and Alexa 647 anti-CD3ε. Samples were washed and fixed immediately with 2% paraformaldehyde. Images were acquired on a Zeiss LSM 510 META (Zeiss, Oberkochen, Germany) laser scanning confocal at 100x magnification at the Johns Hopkins School of Medicine Microscopy Facility. CD3ε cluster size was determined using a particle-detection algorithm written in ImageJ (National Institutes of Health) using the built-in Particle Analyzer. Particle auto-fluorescense for cells bound to particles was removed manually.