Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Summary

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Abstract

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

Introduction

Invariant Natural killer T cells (iNKT cells) are innate-like T lymphocytes that express a semi-invariant αβ T cell receptor (TCR), formed in mice by an invariant Vα14-Jα18 chain paired with a limited set of diverse Vβ chains¹, which is specific for lipid antigens presented by the MHC class I-related molecule CD1d². iNKT cells undergo an agonist selection program resulting in the acquisition of an activated/innate effector phenotype already in the thymus, which occurs through several maturation stages^3,4, producing a CD4⁺ and a CD4^- subset. Through this program, iNKT cells acquire distinct T helper (T_H) effector phenotypes, namely T_H1 (iNKT1), T_H2 (iNKT2) and T_H17 (iNKT17), identifiable by the expression of the transcription factors T-bet, GATA3, PLZF, and RORγt, respectively⁵. iNKT cells recognize a range of microbial lipids but are also self-reactive against endogenous lipids that are upregulated in the context of pathological situations of cell stress and tissue damage, such as cancer and autoimmunity². Upon activation, iNKT cells modulate the functions of other innate and adaptive immune effector cells via direct contact and cytokine production².

The investigations of iNKT cells have been facilitated by mouse models, including CD1d-deficient or Jα18-deficient mice, and by the production of antigen-loaded CD1d tetramers plus the generation of monoclonal antibodies (mAbs) specific for the human semi-invariant TCR. However, the generation of primary mouse iNKT cell line has proved difficult. To better characterize the antitumor functions of iNKT cells and to utilize them for adoptive cell therapy, we set up a protocol to purify and expand splenic iNKT cells of iVα14-Jα18 transgenic mice (iVα14Tg)⁶, in which iNKT cells are 30 times more frequent than in wild type mice.

Expanded iNKT cells can be exploited for in vitro assays, and in vivo upon transfer back into mice. In this setting, for example, we have shown their potent anti-tumor effects⁷. Moreover, in vitro expanded iNKT cells are amenable to functional modification via gene transfer or editing prior to their injection in vivo⁸, allowing insightful functional analysis of molecular pathways, as well as paving the way for advanced cell therapies.

Protocol

Procedures described here were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (no. 1048) at the San Raffaele Scientific Institute.

NOTE: All the procedures must be performed under sterile conditions. All the reagents used are listed in the Table of Materials.

1. Spleen processing

1. Euthanize iVα14-Jα18 mice by inhalation of CO₂ according to the institutional policy.
   NOTE: iVα14-Jα18 mice must be 8 weeks old or older. To avoid rejection of the cells from the in vivo transfer of the cells, consider that cells isolated from female mice can be adoptively transferred both in male and female recipients, whereas cells isolated from male mice can be transferred only in male recipients. For in vitro experiments, no gender bias must be considered. More mice can be pooled in order to obtain more cells.
2. Dissect the mouse spleen and smash it through a 70 nm cell strainer to obtain a single-cell suspension in 10 mL of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS).
3. Centrifuge at 300 x g for 5 min.
4. Remove the supernatant by inversion and process with erythrocyte lysis buffer. Resuspend the cell pellet with 1 mL of sterile ACK (Ammonium-Chloride-Potassium) Lysing Buffer, incubate for 3 min at room temperature, and block with 5 mL of PBS with 2% FBS. Centrifuge at 300 x g for 5 min.
   NOTE: ACK is commercially available; it consists of a solution of 0.15 M NH₄Cl, 10 mM KHCO₃, and 0.1 mM Na₂EDTA (ethylenediaminetetraacetic acid) dissolved in bidistilled H₂O, pH 7.2-7.4. If homemade, sterilize by filtration with a 0.22 µm filter.
5. Remove the supernatant by inversion. Resuspend the cell pellet in 3 mL of PBS with 2% FBS and remove fat residues by pipetting. If needed, pool the cells coming from different mice.
6. Count the cells and keep 50 µL for FACS analysis.

2. T cell enrichment

NOTE: For the enrichment steps, work fast, keep the cells cold and use solutions pre-cooled at 4 °C overnight and then kept on ice

1. Centrifuge at 300 x g for 5 min.
2. Resuspend all the cells in the appropriate amount of PBS with 2% FBS (500 µL for 10⁷ cells) and Fc blocker (2.5 µL x 10⁷ cells); incubate for 15 min at room temperature (RT).
3. Wash with 1-2 mL of MACS separation buffer (MB) per 10⁷ total cells and centrifuge at 300 x g for 10 min.
   NOTE: MACS buffer is commercially available. It consists of PBS pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA. If homemade, sterilize by filtration with a 0.22 µm filter.
4. Remove the supernatant by inversion and stain the cells with CD19-FITC and H2(IA^b)-FITC (use 5 µL x 10⁷ cells in 100 µL of MB). Mix well and incubate for 15 min in the dark at 4-8 °C.
5. Wash cells by adding 1−2 mL of MB per 10⁷ cells and centrifuge at 300 x g for 10 min.
6. Pipette off the supernatant completely and resuspend the cell pellet in 90 µL of MB per 10⁷ total cells. Add 10 µL of Anti-FITC MicroBeads per 10⁷ total cells. Mix well and incubate for 15 min in the dark at 4-8 °C.
7. Wash the cells by adding 1−2 mL of MB per 10⁷ cells and centrifuge at 300 x g for 10 min.
8. Pipette off the supernatant completely and resuspend up to 1.25 x 10⁸ cell in 500 µL of MB.
9. Place a LD column in the magnetic field of the MACS separator to proceed to the depletion. To avoid clogging, apply a pre-separation filter on the LD column and rinse with 2 mL of MB.
10. When the column reservoir is empty, apply the cell suspension onto the filter. Collect the unlabeled cells that pass through the column.
11. Wash 3 times with 1 mL of MB, only when the column reservoir is empty. Collect the total effluent, which will be enriched in T cells, and count the cells. Always keep 50 µL for FACS analysis.

3. iNKT cell enrichment

1. Centrifuge at 300 x g for 5 min and remove the supernatant by inversion.
2. Stain the cells with CD1d-tetramer-PE (mouse PBS57-CD1d-tetramer), according to the antibody titration in 50 µL of MB per 10⁶ cells. Mix well and incubate for 30 min in the dark on ice.
   NOTE: The procedure can also be performed with APC-labelled mCD1d tetramers and anti-APC beads; adjusting the fluorochromes used in the following staining accordingly. In the present protocol we used mouse PBS-57-CD1d-tetramers provided by NIH. αgalactosylceramide (αGal-Cer) is the prototypical antigen recognized by iNKT cells; PBS-57 is an analogue of αGal-Cer with improved solubility⁹; the NIH Tetramer Facility provides PBS-57 ligand complexed to CD1d tetramers. However, other CD1d dimers/tetramers/dextramers are commercially available and can be loaded with a lipidic antigen as αGal-Cer. We envisage the possibility to adjust the protocol for their use.
3. Wash the cells by adding 1−2 mL of MB per 10⁷ cells and centrifuge at 300 x g for 10 min.
4. Pipette off the supernatant completely and resuspend the cell pellet in 80 µL of MB per 10⁷ total cells. Add 20 µL of Anti-PE MicroBeads per 10⁷ total cells. Mix well and incubate for 15 min in the dark at 4-8 °C.
5. Wash cells by adding 1−2 mL of MB per 10⁷ cells and centrifuge at 300 x g for 10 min.
6. Pipette off the supernatant completely and resuspend up to 10⁸ cells in 500 µL of MB. Otherwise if cells exceed 10⁸, adjust the volume accordingly.
7. According to the cell count, place a LS (up to 10⁸) or MS (up to 10⁷) column in the magnetic field of the MACS separator. Rinse the column with MB (3 mL for LS, 500 µL for MS).
8. Apply the cell suspension onto the column.
9. Collect unlabeled cells that pass through. Wash the column 3 times by adding the appropriate amount of MB (3 x 3 mL for LS column, 3 x 500 µL for MS column) only when the column reservoir is empty. The total effluent is the negative fraction.
10. Remove the column from the magnetic field and place it on a new collection tube.
11. Pipette MB onto the column (5 mL for LS column or 1 mL for MS column); push the provided plunger into the column and flush out the positive fraction (enriched in iNKT cells).
12. To further increase the iNKT cell recovery, centrifuge the negative fraction at 300 x g for 10 min and repeat steps 3.6-3.7 with a new LS or MS column. Pool the positive fractions and determine cell count. Keep 50 µL of both positive and negative fractions for FACS analysis.
13. Check the Purification steps by FACS analysis. Samples include: spleen ex-vivo, T cell enriched fraction, iNKT positive fraction and iNKT negative fraction. Stain the cells with: CD19-FITC, IAb-FITC, CD1d-tetramer-PE, TCRβ-APC and DAPI.
    NOTE: The expected recovery from one iVα14-Jα18 mouse is 2x10⁶ iNKT cells .

4. iNKT cell activation and expansion

1. Activate purified iNKT cells with mouse T activator anti-CD3/CD28 magnetic beads in a 1:1 ratio.
   1. Centrifuge the iNKT cell positive fraction at 300 x g for 5 min.
   2. Meanwhile transfer the appropriate volume of anti-CD3/CD28 magnetic beads to a tube and add an equal volume of PBS, vortex for 5 seconds. Place the tube on a magnet for 1 minute and discard the supernatant.
   3. Remove the tube from the magnet and resuspend the washed magnetic beads in the proper volume of complete RPMI (RPMI 1640 medium, 10% heat-inactivated FCS, 1 mM sodium pyruvate, 1% Non-essential amino acids, 10 U/ml penicyllin and streptomycin, 50 μM β-Mercaptoethanol) to have 5 x 10⁵ iNKT cells in 1 mL. Use this suspension to resuspend the centrifuged iNKT positive fraction.
2. Plate 1 mL of the cell suspension (5 x 10⁵ iNKT cells) and anti-CD3/CD28 magnetic beads in a 48 well plate with 20 U/mL IL-2 and incubate at 37 °C.
3. After 5 days, add 10 ng/mL IL-7.
4. Split the cells 1:2 when they reach 80-90% confluence, always add 20U/mL IL-2 and 10 ng/mL IL-7. In these conditions, iNKT cells can be expanded for up to 15 days.

Results

The protocol described in this manuscript enables to enrich iNKT cells from the spleen of iVa14-Ja18 transgenic mice through an immunomagnetic separation process summarized in Figure 1A. Total spleen T cells are first negatively selected by depleting B cells and monocytes, followed by iNKT cell positive immunomagnetic sorting with PBS-57 lipid antigen loaded CD1d tetramers, that enable to specifically stain only iNKT cells. This protocol yields about 2 x 10⁶ of 95-98% pure iNKT ce...

Discussion

Here we show a reproducible and feasible protocol to obtain millions of ready-to-use iNKT cells. Due to the paucity of these cells in vivo, a method to expand them was highly needed. The protocol we propose requires neither a particular instrumentation nor a high number of mice. We exploited iVα14-Jα18 transgenic mice on purpose to reduce the number of mice needed for the procedure.

Another successful protocol for iNKT cell expansion from iVα14-Jα18 transgenic mice is avail...

Materials

Name	Company	Catalog Number	Comments
Ammonium-Chloride-Potassium (ACK) solution	in house		0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2-7.4
anti-FITC Microbeads	Miltenyi Biotec	130-048-701
anti-PE Microbeads	Miltenyi Biotec	130-048-801
Brefeldin A	Sigma	B6542
CD19 -FITC	Biolegend	115506	clone 6D5
CD1d-tetramer -PE	NIH tetramer core facility		mouse PBS57-Cd1d-tetramers
CD4 -PeCy7	Biolegend	100528	clone RM4-5
Fc blocker	BD Bioscience	553142
Fetal Bovine Serum (FBS)	Euroclone	ECS0186L	heat-inactivated and filtered .22 before use
FOXP3 Transcription factor staining buffer	eBioscience	00-5523-00
H2 (IAb) -FITC	Biolegend	114406	clone AF6-120.1
hrIL-2	Chiron Corp
Ionomycin	Sigma	I0634
LD Columns	Miltenyi Biotec	130-042-901
LS Columns	Miltenyi Biotec	130-042-401
MACS buffer (MB)	in house		0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 2Mm EDTA
MS Columns	Miltenyi Biotec	130-042-201
Non-essential amino acids	Gibco	11140-035
Penicillin and streptomycin (Pen-Strep)	Lonza	15140-122
PermWash	BD Bioscience	51-2091KZ
PFA	Sigma	P6148
Phosphate buffered saline (PBS)	EuroClone	ECB4004L
PMA	Sigma	P1585
Pre-Separation Filters (30 µm)	Miltenyi Biotec	130-041-407
Recombinat Mouse IL-7	R&D System	407-ML-025
RPMI 1640 with glutamax	Gibco	61870-010
sodium pyruvate	Gibco	11360-039
TCRβ -APC	Biolegend	109212	clone H57-597
αCD3CD28 mouse T activator Dynabeads	Gibco	11452D
β-mercaptoethanol	Gibco	31350010