Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

Abstract

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Figure 1:. Schematic of the process of antigen-specific enrichment and expansion using nanoparticle artificial antigen-presenting cells.

First, complete a no-touch CD8+ T cell isolation. Then, add nanoparticle aAPCs to the CD8+ T cells. Enrich with a magnetic field, culture, and stimulate with aAPCs. Finally, detect enriched and expanded antigen-specific CD8+ T cells by flow cytometry.

Figure 2:. Schematic for conjugating peptide-loaded MHC-Ig and co-stimulatory molecules to the surface of amine-coated magnetic particles.

Briefly, Sulfo-SMCC crosslinker is used to functionalize the magnetic particle surface with maleimide functional groups. MHC-Ig and co-stimulatory molecules are simultaneously functionalized with Traut’s reagents to produce thiol functional groups. The activated particles and protein signals are reacted together and then washed to produce antigen-specific artificial antigen-presenting cell magnetic nanoparticles. This figure has been modified from supplemental material of our laboratory’s publication in Nano Letters.

Figure 3:. Schematic for conjugating peptide-loaded MHC-Ig and co-stimulatory molecules to the surface of NHS-coated magnetic particles.

Briefly, the NHS-coated particles are reacted together with peptide-loaded MHC-Ig and co-stimulatory molecules and then washed to produce antigen-specific artificial antigen-presenting cell magnetic nanoparticles. This figure has been modified from supplemental material of our laboratory’s publication in Nano Letters.

Figure 4:. Schematic for conjugating peptide-loaded MHC-Ig and co-stimulatory molecules to the surface of anti-biotin-coated magnetic particles. MHC-Ig and co-stimulatory molecules are functionalized with NHS-biotin to produce biotin functional groups.

Then the anti-biotin-coated particles are reacted together with the functionalized peptide-loaded MHC-Ig and co-stimulatory molecules. Afterwards, these particles are washed to produce antigen-specific artificial antigen-presenting cell magnetic nanoparticles. This figure has been modified from supplemental material of our laboratory’s publication in Nano Letters.

Figure 5:. Conjugation efficiency is critical for the enrichment and expansion of antigen-specific T cells.

(a) Representative data for conjugation efficiency with the three conjugation methods to three different base magnetic particles described in the paper: amine-coated particles, NHS-coated particles, and anti-biotin-coated particles. Each data point represents a different particle preparation technique and error bars represent S.E.M. (b) How ligand density affects transgenic CD8+ T cell stimulation, where the ligand density is represented as linear spacing between ligands in nanometers on 600 nm and 50 nm aAPCs (n = 5 and error bars represent S.E.M.). This figure has been modified from our laboratory’s publication in Nano Letters.

Figure 6:. Quality control of aAPC enrichment.

Transgenic Pmel gp100-specific CD8+ T cells were doped in at a 1:1000 ratio into wildtype B6 CD8+ T cells. (a) Fold enrichment was measured using flow cytometry following the enrichment by staining the congenic marker Thy1.1 and CD8. Here was a comparison between signal 1 only particles or Db-Ig loaded with gp100, traditional signal 1 and 2 particles or Db-Ig loaded with gp100 and anti-CD28, and non-cognate signal 1 and 2 particles. (b) Cells were also counted before and after to measure the cell recovery by each of the methods. Data represents three independent experiments and error bars represent S.E.M. Data combined was measured by oneway ANOVA with Tukey’s post-test (*p<0.05, **p<0.01). This figure has been modified from our laboratory’s publication in Nano Letters.

Figure 7:. Quality control of biotinylated dimer.

Gp100-specific CD8+ T cells were isolated from a transgenic Pmel mouse and stained in 100 μL of PBS with three concentrations of biotinylated Db-Ig loaded with gp100 and APC anti-CD8a, using wildtype B6 CD8+ T cells as a negative control.

Figure 8:. Enrichment and expansion of antigen-specific CD8+ T cells.

B6 wildtype CD8+ T cells were enriched with either signal 1 only (Kb-Ig loaded with TRP2) or signal 1 and 2 (Kb-Ig loaded with TRP2 and anti-CD28 conjugated to the surface of the particle). Signal 2 was then added to the enriched fraction of signal 1 only aAPCs and all cells were cultured for 1 days. (a) CD8+ T cells are stained and gated on a live/dead fluorescent stain, then gated CD8+ and KbTRP2+, and compared to a non-cognate Kb-Ig to detect antigen-specific CD8+ T cells. (b) percentage and (c) number of TRP2-specific CD8+ T cells could thus be determined, where higher percentages and numbers of antigen-specific CD8+ T cells could be detected from the signal 1 only enrichment approach (n=1, error bars represent standard deviation, two-tailed paired t test *p < 0.05, **p < 0.01). This figure has been modified from our laboratory’s publication in Nano Letters.

Figure 9:. Enrichment and expansion of human antigen-specific CD8+ T cells.

(a) Representative flow cytometry plots on day 0 before the enrichment and day 7 show the dramatic effects of enriching and expanding antigen-specific CD8+ T cells from healthy donors with traditional nanoparticle aAPCs where A2-Ig loaded with NY-ESO1 and A2-Ig loaded with MART1 antigens are shown. (b) This generates high percentages (~10–20%) and numbers (0.5–1 × 10⁶) of antigen-specific CD8+ T cells by day 7 (n = 3 from independent donors, error bars represent S.E.M.). This figure has been modified from our laboratory’s publication in ACS Nano.