Development and Function of Invariant Natural Killer T Cells Producing TH2- and TH17-Cytokines

Four distinct subsets of invariant natural killer T (NKT) cells are shown to differentiate in the thymus, then migrate to peripheral tissues where they retain their phenotypic and functional characteristics

Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype

The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo. Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy

Natural Killer T Cell-Targeted Immunotherapy Mediating Long-term Memory Responses and Strong Antitumor Activity

Current tumor therapies, including immunotherapies, focus on passive eradication or at least reduction of the tumor mass. However, cancer patients quite often suffer from tumor relapse or metastasis after such treatments. To overcome these problems, we have developed a natural killer T (NKT) cell-targeted immunotherapy focusing on active engagement of the patient’s immune system, but not directly targeting the tumor cells themselves. NKT cells express an invariant antigen receptor α chain encoded by Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans and recognize glycolipid ligand in conjunction with a monomorphic CD1d molecule. The NKT cells play a pivotal role in the orchestration of antitumor immune responses by mediating adjuvant effects that activate various antitumor effector cells of both innate and adaptive immune systems and also aid in establishing a long-term memory response. Here, we established NKT cell-targeted therapy using a newly discovered NKT cell glycolipid ligand, RK, which has a stronger capacity to stimulate both human and mouse NKT cells compared to previous NKT cell ligand. Moreover, RK mediates strong adjuvant effects in activating various effector cell types and establishes long-term memory responses, resulting in the continuous attack on the tumor that confers long-lasting and potent antitumor effects. Since the NKT cell ligand presented by the monomorphic CD1d can be used for all humans irrespective of HLA types, and also because NKT cell-targeted therapy does not directly target tumor cells, this therapy can potentially be applied to all cancer patients and any tumor types

Undisturbed TCRα chain joining region usage in newly generated <i>Traj18</i>-deficient mice as revealed by next generation sequencing.

<p>Sequencing of TCRα chain joining region. PCR was carried out to amplify <i>Trav11-Trac</i> transcripts using cDNA prepared from sorted TCRβ^low CD4⁺CD8⁺ double-positive thymocytes from <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- (red bars) and <i>Traj18</i>^-/- (blue bars) mice. Bars depict mean ± SEM percentages of productive <i>Traj</i> gene segment rearrangements, and data are derived from three biologically independent samples per genotype. Numbers in parenthesis indicate the total number of sequences analyzed. The data are from one experiment.</p

Newly generated <i>Traj18</i>-deficient mice lack Vα14 NKT cells.

<p>(A) Flow cytometry profiles of thymocytes, splenocytes and liver mononuclear cells from WT, <i>Traj18</i>^-/- and <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- mice. Unloaded CD1d dimer staining was used as a staining control. Numbers depict percentage of αGC/CD1d dimer⁺ TCRβ⁺ NKT cells among viable CD8^-B220^- gated lymphocytes. The data are representative of three independent experiments. (B) <i>In vivo</i> cytokine production by NKT cells upon systemic activation with αGalCer administration. WT or <i>Cd1d1</i>^-/-<i>Cd1d2</i>^-/- or <i>Traj18</i>^-/- mice were injected intravenously with 2 μg of αGalCer and blood plasma were collected after either 3 h and 24 h, and IFN-γ and IL-4 concentrations were measured using cytokine beads assay. Bars depict mean ± SEM of <i>n</i> = 3 mice per genotype analyzed. Data are representative of three experiments.</p

Generation of novel <i>Traj18</i>-deficient mice.

<p>Schematic representation of a <i>Traj18</i> region targeting construct, <i>Traj18</i> region before and after homologous recombination, and the genomic locus after FLP- and Cre-mediated deletions of the neomycin resistance gene and <i>Traj18</i>, respectively.</p

A validation of the adjuvant effect of Vα14 NKT cells using <i>Traj18</i>-deficient mice.

<p>(A) NKT cell-mediated adjuvant effect on the expansion of antigen-specific CD8 T cells. WT and <i>Traj18</i>^-/- mice were immunized with OVA antigen and αGalCer on day 0, and splenocytes were analyzed on day 7. Numbers on FACS plots represent percentage of OVA-tetramer positive cells among viable CD8 T cells. (B) Cell percentages and (C) numbers of OVA-tetramer positive cells gated as shown in A. Bars depict mean ± SEM for <i>n</i> = 9 mice per group. (D) NKT cell-mediated adjuvant effect on the activation of antigen-specific CD8 T cells. WT and <i>Traj18</i>^-/- mice were immunized with OVA antigen and αGalCer on day 0, and splenocytes were harvested on day 7. Cells then were cultured <i>in vitro</i> with or without OVA_257-264 peptide for 6 h in the presence of GolgiPlug Protein Transport Inhibitor, and were stained with an IFN-γ mAb using Cytofix/Cytoperm kit. Numbers on FACS plots represent percentage of IFN-γ positive cells among CD8 T cells. (E) Percentages and (F) numbers of IFN-γ positive cells shown in D. Bars graphs depict mean ± SEM for <i>n</i> = 5 mice per group. All data shown are representative from three independent experiments. ****, <i>P</i> < 0.0001 using unpaired <i>t</i> test.</p

Normal development of MAIT cells with an invariant Vα19Jα33 in <i>Traj18</i>-deficient mice.

<p>(A) Sorting strategy of αGC/CD1d^- TCRβ⁺ lung T lymphocytes from WT, <i>Traj18</i>^<i>-/-</i> and previously generated <i>Jα18</i>^<i>-/-</i> mice. Numbers on FACS plots depict percentage of gated cells among viable 7-AAD^- B220^- lung lymphocytes. (B) Relative expression of Vα19Jα33 mRNA by real-time quantitative RT-PCR in sorted lung cells shown in (A). Gene expression was normalized using <i>Trac</i> as internal control. Bars depict mean ± SEM, n.s., not significant using unpaired <i>t</i> test. All data are representative of three independent experiments with a combined total of three mice per genotype.</p

<i>i</i>NKT cell subtypes in the periphery.

<p>(A–C) FACS profile of peripheral <i>i</i>NKT cells in B6 mice. α-GalCer/CD1d dimer⁺ TCRβ⁺<i>i</i>NKT cells (A) and <i>i</i>NKT subtypes based on the expression of CD44 and NK1.1 (B) or CD4 and IL-17RB (C) in spleen, liver, BM, lung, inguinal LN, and mesenteric LN from B6 or <i>Il17rb</i>^−/− mice. Numbers indicate percentage of total mononuclear cells (A) and <i>i</i>NKT cells (B, C). (D) Number of cells of each <i>i</i>NKT subtype based on the expression of IL-17RB and CD4 in thymus and periphery of B6 and BALB/c mice. Cell numbers were calculated based on the results from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001255#pbio-1001255-g004" target="_blank">Figures 4A, 4C</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001255#pbio.1001255.s008" target="_blank">S8A</a>, and S8B. IL-17RB⁺<i>i</i>NKT cells were mainly localized in spleen, lung, inguinal LN, and mesenteric LN, whereas hardly any were observed in liver and BM. One representative experiment of three is shown.</p

Function of <i>i</i>NKT cell subtypes in the spleen.

<p>(A) Global gene expression profiles in <i>i</i>NKT subtypes in the thymus and spleen. Tree view representation of clustering analysis among the four <i>i</i>NKT subtypes in thymus and spleen from B6 and BALB/c. The values represent coefficients between the indicated panels. <i>r</i>²>0.95 in red, 0.85<<i>r</i>²<0.95 in orange, and <i>r</i>²<0.85 in blue. One representative experiment of three is shown. (B) Plasticity and stability of <i>i</i>NKT subtypes. The four <i>i</i>NKT cell subtypes in the thymus were sorted and each subtype (5×10⁵) was i.v. transferred into independent <i>Jα18</i>^−/− mice (<i>n</i> = 3). 10 d after transfer, α-GalCer/CD1d dimer⁺ TCRβ⁺ cells in spleen were analyzed by FACS for the expression of IL-17RB and CD4. Representative data from three experiments are shown. (C–F) In vitro cytokine production by splenic <i>i</i>NKT cell subtypes (red, CD4⁻ IL-17RB⁺; orange, CD4⁺ IL-17RB⁺; blue, CD4⁻ IL-17RB⁻; green, CD4⁺ IL-17RB⁻). Sorted splenic <i>i</i>NKT subtypes (5×10⁴ cells/100 µL) were co-cultured with BM-DCs (5×10³/100 µL) for 48 h in the presence of α-GalCer (100 ng/µL) (C), IL-12 (10 ng/µL) (D), IL-23 (10 ng/µL) (E), and IL-25 (10 ng/µL) (F). Levels of IFN-γ, IL-4, IL-9, IL-10, IL-13, IL-17A, and IL-22 in the supernatants were analyzed by ELISA or CBA. Data are mean ± SD of triplicate wells. One representative experiment of three is shown.</p