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.
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 stages3,4, producing a CD4+ and a CD4- subset. Through this program, iNKT cells acquire distinct T helper (TH) effector phenotypes, namely TH1 (iNKT1), TH2 (iNKT2) and TH17 (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.
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
- 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.
- 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).
- Centrifuge at 300 x g for 5 min.
- 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.
- 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.
- 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
- Centrifuge at 300 x g for 5 min.
- 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).
- 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.
- Remove the supernatant by inversion and stain the cells with CD19-FITC and H2(IAb)-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.
- Wash cells by adding 1−2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.
- 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.
- Wash the cells by adding 1−2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.
- Pipette off the supernatant completely and resuspend up to 1.25 x 108 cell in 500 µL of MB.
- 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.
- When the column reservoir is empty, apply the cell suspension onto the filter. Collect the unlabeled cells that pass through the column.
- 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
- Centrifuge at 300 x g for 5 min and remove the supernatant by inversion.
- 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.
- Wash the cells by adding 1−2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.
- 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.
- Wash cells by adding 1−2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.
- 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.
- 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).
- Apply the cell suspension onto the column.
- 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.
- Remove the column from the magnetic field and place it on a new collection tube.
- 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).
- 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.
- 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
- Activate purified iNKT cells with mouse T activator anti-CD3/CD28 magnetic beads in a 1:1 ratio.
- Centrifuge the iNKT cell positive fraction at 300 x g for 5 min.
- 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.
- 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.
- 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.
- After 5 days, add 10 ng/mL IL-7.
- 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.
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 cells from the spleen of a single iVa14-Ja18 Tg mouse. No or really few iNKT cells can be detected in the negative fraction (Figure 1B).
After enrichment, iVa14 iNKT cells can be expanded with anti-CD3/CD28 beads plus IL-2 and IL-7 (Figure 2A), resulting in 30-fold expansion on average by day +14 of the culture as shown in Figure 2B.
Figure 3A shows the iNKT cell purity along with the expansion in vitro and the expression of the CD4 molecule. We observed a diminishment in the percentage of TCRβ+ CD1d-tetramer+ double positive cells: the strong activation with anti-CD3/CD28 beads is inducing the downregulation of the iNKT cell TCR expression on the cell surface, and a double negative population is appearing. The majority of expanded iNKT cells were CD4-. Figure 3B shows a characterization of the expression of lineage-specific transcription factors PLZF and RORγt on enriched iNKT cells at day 0 and 14 days after the expansion. This staining enables to identify the NKT1 (PLZFlow RORγt-), NKT2 (PLZFhigh RORγt-), and NKT17 (PLZFint RORγt+) phenotypes. Being mostly NKT1 and NKT2, the enriched iNKT cells show a TH0-like effector phenotype. This phenotype is conserved after 14 days of expansion as confirmed by the secretion of both IFN-γ and IL-4 after PMA/Ionomycin stimulation shown in Figure 3C.
Figure 1: iNKT cell enrichment. A) Schematic representation of the immunomagnetic separation protocol. B) Flow cytometry analysis of each enrichment step. Percentage of T cell frequencies are shown in the upper plots, gated on viable lymphocytes. While percentage of iNKT cell frequencies along each step are shown in the lower plots, gated on viable CD19- TCRβ+ lymphocytes. Staining on viable CD19- TCRβ+ lymphocytes with unloaded CD1d tetramer allow to correctly draw the iNKT cell gate. One representative experiment is shown. Please click here to view a larger version of this figure.
Figure 2: iNKT cell in vitro expansion. A) iNKT cell counts along iNKT cell expansion. Three representative and independent experiments are shown. B) Fold increase in iNKT cell number at day 7 and 14 after purification and activation. Means ± SD are shown. Please click here to view a larger version of this figure.
Figure 3: Expanded iNKT cell characterization. A) Flow cytometry analysis of iNKT cell percentage and CD4 expression along the expansion period. Upper plots are gated on viable lymphocytes. Lower plots are gated on iNKT cells (viable CD1d-tetramer+ TCRβ+ lymphocytes). B) Phenotypic characterization of enriched (day 0) and expanded (day 14) iNKT cells. Plots are gated on iNKT cells (viable CD1d-tetramer+ TCRβ+ lymphocytes). Cells were intranuclearly stained for transcription factors with the Foxp3 Transcription Factor Staining Buffer Set. NKT1 (PLZFlow RORγt-), NKT2 (PLZFhigh RORγt-), and NKT17 (PLZFint RORγt+) subsets were identified, frequencies of each subset are shown in percentage. C) Cytokine production by expanded iNKT cells at day 14. Plots are gated on iNKT cells (viable CD1d-tetramer+ TCRβ+ lymphocytes). Cells were stimulated for 4 hours with PMA 25 ng/mL/Ionomycin 1 µg/mL, in the presence for the last 2 hours of Brefeldin A 10 µg/mL. Cells were then fixed with PFA 2%, permeabilized with Permwash and then intracellularly stained for cytokine production. Gating strategy was set on the non-activated control, left panel. One representative experiment is shown. Please click here to view a larger version of this figure.
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 available in the literature10. This protocol involves the generation, 6 days prior to iNKT cell purification, of fresh bone marrow-derived dendritic cells, then loaded with α-galactosylceramide and irradiated, plus IL-2 and IL-7. We consider the reduction of the number of the mice involved in the procedure a great advantage of the protocol. It is also time-sparing, since the setting up of the cell culture lasts a single day instead of a week. A possible limitation of the reproducibility of the current protocol could be the availability of iVα14-Jα18 transgenic mice, that are however commercially available. In absence of these mice, we envisage the possibility of using a large number of WT mice, but the protocol needs to be set up accordingly due to the paucity of iNKT cells in WT mice.
During the cell culture, we usually check the purity and the phenotype of expanded iNKT cells. The decrease in the percentage of TCRβ+ CD1d-tetramer+ double positive cells (Figure 3A) can be explained by a natural downregulation of the invariant NKT cell TCR from the cell surface after activation. Moreover, the majority of expanded iNKT cells did not express CD4 (Figure 3A): this may represent an advantage in the context of an adoptive cell therapy, since CD4- iNKT cells were found to be the most effective in controlling tumor progression11. Moreover, the observed TH0-like effector phenotype (Figure 3C) is entirely coherent with that observed in human iNKT cells after in vitro expansion and restimulation8,12,13,14,15. The expanded cells are highly reactive in vivo and in vitro, thus useful in contexts of iNKT cell-based adoptive immunotherapies. Adoptive transfer of unmanipulated or expanded iNKT cells prevents or ameliorates acute Graft-Versus-Host Disease (aGVHD) leaving unaltered the Graft-Versus-Leukaemia effect16,17,18,19. Adoptively transferred human iNKT-cells expanded in vitro with αGal-Cer alleviate xenogeneic aGVHD and this effect is mediated by CD4- but not CD4+ cells20. Moreover, given that iNKT cells do not cause aGVHD, they constitute the ideal cells for CAR immunotherapy without need for deletion of their TCR and proved to have prolonged antitumor activity in vivo8,15. iNKT cells are currently exploited in on-going and concluded clinical trials21,22,23,24.
In conclusion, the described protocol is fast, straightforward and allows a 30x increase in the number of iNKT cells recovered from a mouse spleen (Figure 3B). These cells can be easily exploited for in vitro recognition assays, co-culture systems, or adoptive cell therapy in preclinical studies. iNKT cells indeed, play a critical role in tumor immune surveillance, infectious diseases and autoimmunity. In these contexts, iNKT cells can represent a powerful tool, being an appealing alternative to conventional T cells devoid of the MHC restriction. The rapid generation of large amounts of these cells and the possibility to further manipulate them in vitro can lead to the development of unprecedented therapeutical strategies.
The authors have nothing to disclose.
We thank Paolo Dellabona and Giulia Casorati for scientific support and critical reading of the manuscript. We also thank the NIH Tetramer Core Facility for mouse CD1d tetramer. The study was funded by Fondazione Cariplo Grant 2018-0366 (to M.F.) and Italian Association for Cancer Research (AIRC) fellowship 2019-22604 (to G.D.).
|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|
|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|
|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|
|Phosphate buffered saline (PBS)||EuroClone||ECB4004L|
|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|
|TCRβ -APC||Biolegend||109212||clone H57-597|
|αCD3CD28 mouse T activator Dynabeads||Gibco||11452D|
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