Ex Vivo Expansion of Th2-Polarizing Immunotherapeutic iNKT Cells from Human Peripheral Blood

Abstract

iNKT cells, classified as innate lymphocytes with invariant TCRs, have been highlighted as a putative, “off-the-shelf” cellular immunotherapeutic strategy for the treatment of malignant and non-malignant diseases. However, their paucity in human blood limits their immunotherapeutic applications. Herein we describe a rigorously optimized 21-day ex vivo expansion method to achieve log-fold increases in immunotherapeutic human iNKT cells.

1. Introduction

Human invariant Natural Killer T (iNKT) cells are a specialized subset of αβ T lymphocytes that express an invariant TCR α-chain(Vα24Jα18), paired with the TCR β-chain, Vβ11 (1—3). Unlike conventional αβ T lymphocytes, iNKT cells neither recognize peptide antigens nor are restricted by conventional polymorphic MHC/HLA molecules; instead, they recognize glycolipid antigens, such as the synthetic glycolipid, α-galactosylceramide (α-GalCer), presented on the non-polymorphic glycoprotein molecule CD1d (4—7). Once stimulated by CD1d-presented glycolipids, iNKT cells can rapidly secrete both Th1 and Th2 cytokines. iNKT cells have been extensively studied for their potent role in immunoregulation, given their ability to promote direct and indirect tolerogenic effects (7—11). After allogeneic transplantation, for example, iNKT cells have been found to regulate graft-versus-host disease (GvHD), while simultaneously maintaining potent graft-versus-tumor (GVT) effects. This separation of undesirable GvHD from the salutary GVT driven by allogeneic T cells is considered the “holy grail” of hematopoietic stem cell transplantation (HSCT) (12—15). However, the advancement of iNKT cellular immunotherapies is significantly hindered by their low frequency in peripheral blood(< 0.01%). To address this need, we have developed a highly reproducible and robust 21-day method for the ex vivo expansion of human peripheral blood iNKT cells. Our protocol incorporates concepts from conventional 3-step signaling mechanisms in T cell activation (16) tailored specifically for optimal iNKT cell expansion, namely, the use of α-GalCer (signal 1: antigen-iTCR engagement) presented on PBMC-derived APCs (signal 2: co-stimulation) and supplemented with recombinant human (rh)IL-2 and (rh)IL-7 (signal 3: cytokines/growth factors) to achieve a striking 320-fold (median) increase of iNKT cells from day 0 to day 21 (17). Additionally, we show via the ⁵¹Cr release assay, that a single-step signal 1/signal 2 activation of expanded and sorted iNKT cells can significantly enhance their cytotoxicity against the CD1d-expressing Jurkat (T-ALL) cell line as compared to expansion alone.

2. Materials

2.1. iNKT Cell Expansion and Culture

1. Human PBMCs: may be purchased or isolated from single-donor blood products by Ficoll-Plaque Plus^® density gradient (see Note 1).

2. iNKT Media: RPMI 1640^® w/ L-glutamine, 10mM HEPES, 0.02 mg/mL gentamicin, 10% human AB serum (see Note 2). Store at 4°C and warm to 37°C prior to use in culture.

3. Sterile 0.45μm CA membrane vacuum filtration unit (see Note 3)

4. Cytokines and growth factors: 100 U/mL rhIL-2, 0.4 ng/mL or 4 ng/mL rhIL-7 and 100 ng/mL α-galactosylceramide (α-GalCer) (see Note 4)

5. Stimulating antibody: 1μg/mL anti-CD3 monoclonal antibody (MoAb)

6. Irradiator (see Note 5)

7. 0.1% Trypan Blue

8. Hemocytometer or automated cell counter

9. T-75 sterile cell culture flasks (see Note 6)

2.2. Flow Cytometry and Cell Sorting

1. Cell Sorter

2. Cell Analyzer

3. Antibodies (see Tables 1 and 2)

4. FACS Buffer: 1X PBS (Ca²⁺, Mg²⁺ free), 1% human AB serum (see Note 2), 0.5mM EDTA, 0.1% NaN₃

5. Sort Buffer: 1X PBS (Ca²⁺, Mg²⁺ free), 2% human AB serum (see Note 2)

6. 2% Paraformaldehyde (PFA)

7. Round bottom polystyrene FACS tubes

8. 15mL sterile conical polypropylene tube

Table 1.

Recommended human iNKT sort panel.

SORT PANEL
Antibody Target	Fluorophore	Clone
CD3	APC	HIT3a
Vα24	PE-Cy7	6B11
Live/Dead	PI or DAPI	--

Table 2.

Recommended Human iNKT flow cytometry panel.

FLOW CYTOMETRY PANEL
Antibody Target	Fluorophore	Clone
CD3	APC	HIT3a
Vα24	PE-Cy7	6B11
Vβ11	PE	REA559
CD4	APC-Cy7	RPA-T4
CD8	eF450	SK1
Live/Dead	BV510 (Ghost 510®)	--

2.3. Functional Assay: Anti-tumor Cytotoxicity

1. Jurkat cell line

2. Jurkat Growth Media: RPMI 1640^® w/ L-glutamine, 10% FBS (see Note 2), 1% Penicilin-Streptomycin. Store at 4°C and warm to 37°C prior to use.

3. Day 21 expanded, sorted human iNKT cells

4. iNKT Media: RPMI 1640^® w/ L-glutamine, 10 mM HEPES, 0.02 mg/mL gentamicin, 10% human AB serum (see Note 2). Store at 4°C and warm to 37°C prior to use.

5. 50 ng/mL α-galactosylceramide, α-GalCer

6. Chromium-51 Radionuclide, ⁵¹Cr (0.1 mCi) (Perkin Elmer)

7. Anti-CD2/3/28 T Cell Activation/Expansion Bead Kit (Miltenyi Biotec)

8. 96-well round bottom plate

9. LumaPlate^™ (ThermoFisher)

10. Liquid scintillation counter

11. Triton-X (SIGMA)

3. Methods

Carry out all procedures at room temperature in a sterile BSL2 biosafety cabinet and follow standard sterile cell culture technique unless otherwise specified.

3.1. iNKT expansion

1. On day 0, plate single-donor human PBMCs in T-75 flasks at a concentration of 2 × 10⁶ cells/mL in iNKT media. (iNKT media should be filtered, warmed to 37°C, and supplemented with 100 U/mL rhIL-2, 0.4 ng/mL rhIL-7 and 100 ng/mL α-GalCer prior to use.) Incubate T-75 flasks for 7 days in 37°C, 5% CO₂ (see Notes 3 and 7).

2. On day 7, stain cells and sort for live CD3⁺, Vα24⁺ (clone 6B11, eBioscience 50-112-3387) iNKT cells using the sort panel shown in Table 1. Collect sorted cells in chilled iNKT media in a 15mL conical polypropylene tube. See representative day 7 pre-sort and post-sort flow plots, Figure 3.

3. Count sorted Day 7 CD3⁺Vα24⁺ iNKT cells using trypan blue exclusion and replate in a T-75 (see Note 6) at a concentration of 1 × 10³ – 5 × 10⁴ iNKT cells/mL of iNKT media with allogeneic PBMC a ratio of sorted iNKT cells to irradiated allogeneic PBMC of 1:50 (see Note 8). Supplement iNKT media with 1μg/mL anti-CD3 MoAb, 100 U/mL rhIL-2 and 4 ng/mL rhIL-7 (see Note 9). Incubate culture flasks, standing flasks upright to enhance cell-cell contact, for an additional 14 days (through day 21), replacing fresh iNKT media supplemented with 100 U/mL rhIL-2 and 4 ng/mL rhIL-7 on days 14 and 21.

   On day 21, iNKT + feeder cells may be cryopreserved for subsequent surface phenotyping and/or functional assays, or they can be stained with antibodies (see Table 2) and CD3⁺Vα24⁺ iNKT cells re-sorted for immediate use. Collect sorted cells in chilled iNKT media in a 15mL conical polypropylene tube and count using trypan blue exclusion. See expansion protocol timeline in Figure 1.

Figure 3.

Representative flow plots showing CD3 and Vα24 expression pre- and post-sort on gated CD3⁺ cells on day 7. Reprinted with permission from (17).

Figure 1.

Human iNKT ex vivo expansion timeline. Reprinted with permission from (17).

3.2. iNKT yield and phenotype assessment by Flow Cytometry

Harvested samples may be further handled under non-sterile conditions. Keep samples on ice at all times to preserve viability and function.

1. Assess and compare iNKT expansion yield and phenotypes by flow cytometry at days 0, 7 (pre-sort), 14 and 21, by harvesting at least 100,000 cells/sample at relevant time points.

2. Wash each sample with 1–2 mL of FACS buffer and centrifuge for 5 minutes at 400×g. Carefully decant supernatant and repeat wash step.

3. Using the panel shown (see Table 2), stain samples and relevant compensation controls for 30 minutes in the dark, on ice.

4. Wash each sample using 100–200 μL of FACS buffer and centrifuge for 5 minutes at 400×g. Carefully decant supernatant and repeat this wash step 2 times further.

5. Fix the stained cells with 100 μL of 2% PFA for 10 minutes in the dark, on ice. Wash with 100 – 200 μL of ice cold FACS buffer and centrifuge for 5 minutes at 800×g. Carefully decant supernatant and repeat this wash step 2 times further.

6. For immediate flow cytometry analyses, resuspend cell pellets in 200 – 300 μL of fresh, ice-cold FACS buffer and transfer to round-bottom FACS tubes to run samples. For future analysis, store cell pellets in the dark at 4°C until time of analysis (see Note 10).

7. Analyze data using FlowJo^® v10. See iNKT expansion yield comparisons between days 0, 7, 14 and 21 in Figure 2 and CD4/CD8 phenotypic comparisons between days 0, 7 and 21 in Figure 4.

Figure 2.

Left panel: median absolute number CD3⁺Vα24⁺ iNKT cells at day 7, day 14, and day 21 stratified by day 7 sort yield categories 10⁴–10⁵ (orange, N = 4), 105–106 (yellow, N = 16), 10⁶–10⁷ (cyan, N =21), and >10⁷ (magenta, N = 6). Right panel: sub analysis of expansion data by day 7 sort categories showing median and IQR ± range absolute number CD3⁺Vα24⁺ iNKT cells at specified time points in the expansion protocol (S, day 7 post-sort). Reprinted with permission from (17).

Figure 4.

Left panel: Median and IQR ± range percentage (top) with representative flow plots (bottom) of CD4 and CD8 expression in gated CD3⁺Vα24⁺ cells on days 0, 7, and 21(N = 36). Right panel: Median and IQR ± range absolute number of CD4⁺, DN, and CD8⁺ CD3⁺Vα24⁺ iNKT cells on days 0, 7, and 21 (left panel; N = 36). P < 0.05 is represented by *. Reprinted with permission from (17).

3.3. iNKT Functional Assay: ⁵¹Cr release anti-tumor cytotoxicity assay

1. In a 24-well plate, stimulate Day 21 iNKT cells (effectors, E) (either freshly sorted or previously sorted, cryopreserved and re-thawed) using anti-CD2/CD3/CD28 loaded beads, control beads or media only (see Note 11). Incubate for 24 hours at 37°C, 5% CO_2.

2. Label Jurkat T-ALL human cell line (targets, T) with 0.1 mCi ⁵¹Cr in 200 μL of pre-warmed Jurkat media. Incubate for 24 hours at 37°C, 5% CO₂ (see Notes 12 and 13).

3. After 24 hours, wash ⁵¹Cr labelled Jurkat cells 3 times with Jurkat media (see Note 13) and plate in triplicate in 96-well round bottom plate at 10⁵ cells/well, either with or without 50 ng/mL α-GalCer.

4. After 24 hours, add stimulated iNKT cell effectors (E) over ⁵¹Cr-labeled Jurkat targets (T) in the 96-well plate holding the target cell number constant and varying the effector numbers to achieve E:T ratios of 8:1 to 0.0625:1. Incubate plates for 18 hours at 37°C in 5% CO₂.

5. After 18 hours of co-culture, centrifuge the 96-well plate at 400×g for 5 minutes and transfer the supernatant to a LumaPlate^™.

6. Place the LumaPlate^™ in a liquid scintillation counter and quantitate beta-emission (see Note 14). See Figure 5 for representative cytotoxicity data across varying E:T ratios.

Figure 5.

Left Panel: Representative histogram showing CD1d expression, normalized to mode, on Jurkat cells. red: isotype control, blue: anti-CD1d. Right Panel: Mean percent cytotoxicity ± SEM of day 21 sorted iNKT effectors (E) against Jurkat targets (T) at the indicated E:T target ratios (x axis) via ⁵¹Cr release assay (N=4). blue circles: iNKT + unloaded beads; blue triangles: iNKT + unloaded beads + 50 ng/mL α-GalCer; red circles: iNKT + anti-CD2/3/28 loaded beads; red triangles: iNKT + anti-CD2/3/28 loaded beads + 50 ng/mL α-GalCer. Reprinted with permission from (17).

4. Notes

1. Two allogeneic donors are needed per single-donor expansion (one is the iNKT expansion product and the other is the allogeneic feeder source, co-cultured with sorted iNKT cells from day 7 onward). Autologous PBMCs may also be used as feeders in place of allogeneic PBMCs. PBMCs may be either freshly isolated prior to expansion or previously cryopreserved and thawed for expansion.

2. Human AB Serum and Fetal Bovine Serum (FBS) must have complement heat-inactivated for 30 minutes at 56° C.

3. We recommend filtering formulated iNKT media through a 0.45 μm CA membrane filtration unit before using for culture.

4. 0.4 ng/mL rhIL-7 (1x) is used for the first 7 days of expansion, prior to the first sort. 4 ng/mL rhIL-7 (10x) is used for the remainder of the expansion period, i.e., on day 7/8 post-sort and day 14.

5. For feeder irradiation, either a standard orthovoltage source irradiator or a ¹³⁷Cs (cesium-137) radioisotope source irradiator may be used.

6. Depending on donor-specific iNKT yield variations on day 7 post-sort, T-25 flasks, 6-well, 12-well, 24-well or 48-well plates may be needed to accommodate lower iNKT yields. For example, if day 7 post-sort yield is ~8,000 total iNKT cells (<10⁴), then the number of irradiated allo-PBMCs needed is 50*8000 cells = 400,000 irradiated allo-PBMCs. Thus, the total number of cells to plate is 400,000 allo-PBMCs + 8,000 day 7 iNKTs = 408,000 total cells. Plate these cells in a 48-well plate (1 well), in ~ 400 – 500 μL media.

7. Our data shows that higher iNKT Day 7 yields correlate with higher iNKT yields at the end of 21 days expansion (see Figure 2). Therefore, it is advised to start with at least 2 × 10⁸ PBMCs per donor. Given the recommended PBMC culture concentration of 2 × 10⁶ PBMCs/mL iNKT media, 10mL/flask of PBMC-iNKT media suspension, and a total of 10 T-75 flasks is required for 2 × 10⁸ starting PBMCs.

8. Irradiate the required number of allogeneic or autologous feeder human PBMCs at 5000 cGy. Feeder cells may be either fresh or previously cryopreserved and thawed.

9. 100 U/mL rhIL-2 and 4 ng/mL rhIL-7 may be added as early as day 7, immediately after sorting, or as late as day 10.

10. It is highly recommended to analyze samples within 1 week of staining and fixing.

11. Prepare stimulation beads (loaded with antibodies or unloaded beads) as per manufacturer’s instructions. Stimulate iNKT cells using a 1:2, bead:iNKT cell ratio in 1mL of iNKT media.

12. Harvest Jurkat cell line at 80–90% confluence for use in cytoxicity assay.

13. ⁵¹Cr presents an external dose hazard via its gamma emissions. Users must minimally follow the “Safe Handling Guide: Chromium-51 Handling Precautions” provided by the manufacturer and are encouraged to consult their institutional Radiation Safety specialist to tailor safety precautions to the user’s specific needs and/or applications.

14. Use the following formula to calculate % cytotoxicity from counts per minute (CPM) data collected from liquid scintillation counter:

    $$\frac{\left(CP{M}_{\mathit{\text{experimental}}}-CP{M}_{\mathit{\text{spontaneous}}}\right)}{\left(CP{M}_{\mathit{\text{maximum}}}-CP{M}_{\mathit{\text{spontaneous}}}\right)}\times 100=\%\mathit{\text{ Cytotoxicity}}$$