T Cell Receptor Expression Timing and Signal Strength in the Functional Differentiation of Invariant Natural Killer T Cells

The CD1d-restricted Vα14 invariant NKT (iNKT) cell lineage in mice (Vα24 in humans) represents an evolutionary conserved innate-like immune cell type that recognizes glycolipid antigens. Because of their unique ability to promptly secrete copious amounts of both pro-inflammatory and anti-inflammatory cytokines, typically produced by different T helper cell types, iNKT cells are implicated in the regulation of various pathologic conditions such as infection, allergy, autoimmune disease, maintenance of transplantation tolerance, and cancer. This striking multifaceted role in immune regulation is correlated with the presence of multiple functionally distinct iNKT cell subsets that can be distinguished based on the expression of characteristic surface markers and transcription factors. However, to date it, remains largely unresolved how this puzzling diversity of iNKT cell functional subsets emerges and what factors dictate the type of effector cell differentiation during the thymic differentiation considering the mono-specific nature of their T cell receptor (TCR) and their selecting molecule CD1d. Here, we summarize recent findings focusing on the role of TCR-mediated signaling and discuss possible mechanisms that may influence the sub-lineage choice of iNKT cells

Relationship of Human Personal Characteristics in the three Independent Stratification Systems

Objectives: We aimed to analyze theassociation between the biophysiological characteristics of body constitution and temperament type with Mongolian Traditional Medicine (MTM) constitution type to understand further and integrate the application of these stratification systems in clinical medicine. Methods: Body constitution type was classified using the body-mass index, temperament type was determined using personality test, and dominant MTM constitution type was determined in 287 active blood donors. Age, gender, and ABO blood groups were considered as additional biophysiological characteristics. The correlation between the biophysiological characteristics and the nature of their relationship was investigated using likelihood ratio and receiver operating characteristics analysis. Results: Obesity and body-mass index were related to aging, and donor’s age and Badgan expression rate were shown as a sensitive and specific classifier for obesity state. The relationship between Badgan dominant types of human constitutions practiced in Mongolian Traditional Medicine and melancholic temperament was established. Badgan expression was a sensitive and specific classifier for melancholic and phlegmatic temperament. Conclusions: Stratification of human subjects using the primary types of human constitutions practiced in Mongolian Traditional Medicine may be helpful in clinical research and application

Identification of CD4−CD8− Double-Negative Natural Killer T Cell Precursors in the Thymus

BACKGROUND: It is well known that CD1d-restricted Valpha14 invariant natural killer T (NKT) cells are derived from cells in the CD4(+)CD8(+) double-positive (DP) population in the thymus. However, the developmental progression of NKT cells in the earlier stages remains unclear, and the possible existence of NKT cell presursors in the earlier stages than DP stage remains to be tested. PRINCIPAL FINDINGS: Here, we demonstrate that NKT cell precursors that express invariant Valpha14-Jalpha18 transcripts but devoid of surface expression of the invariant Valpha14 receptor are present in the late CD4(-)CD8(-) double-negative (DN)4 stage and have the potential to generate mature NKT cells in both in vivo and in vitro experimental conditions. Moreover, the DN4 population in CD1d knock-out (CD1dKO) mice was similar to those with an NKT cell potential in wild-type (WT) C57BL/6 (B6) mice, but failed to develop into NKT cells in vitro. However, these precursors could develop into NKT cells when co-cultured with normal thymocytes or in an in vivo experimental setting, indicating that functional NKT cell precursors are present in CD1dKO mice. CONCLUSIONS: Together, these results demonstrate that thymic DN4 fraction contains NKT cell precursors. Our findings provide new insights into the early development of NKT cells prior to surface expression of the invariant Valpha14 antigen receptor and suggest the possible alternative developmental pathway of NKT cells

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

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

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