Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to nonpeptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

Vaccine Based on Dendritic Cells Electroporated with an Exogenous Ovalbumin Protein and Pulsed with Invariant Natural Killer T Cell Ligands Effectively Induces Antigen-Specific Antitumor Immunity

(1) Background: Cancer vaccines are administered to induce cytotoxic CD8+ T cells (CTLs) specific for tumor antigens. Invariant natural killer T (iNKT) cells, the specific T cells activated by α-galactosylceramide (α-GalCer), play important roles in this process as they are involved in both innate and adaptive immunity. We developed a new cancer vaccine strategy in which dendritic cells (DCs) were loaded with an exogenous ovalbumin (OVA) protein by electroporation (EP) and pulsed with α-GalCer. (2) Methods: We generated bone marrow-derived DCs from C57BL/6 mice, loaded full-length ovalbumin proteins to the DCs by EP, and pulsed them with α-GalCer (OVA-EP-galDCs). The OVA-EP-galDCs were intravenously administered to C57BL/6 mice as a vaccine. We then investigated subsequent immune responses, such as the induction of iNKT cells, NK cells, intrinsic DCs, and OVA-specific CD8+ T cells, including tissue-resident memory T (TRM) cells. (3) Results: The OVA-EP-galDC vaccine efficiently rejected subcutaneous tumors in a manner primarily dependent on CD8+ T cells. In addition to the OVA-specific CD8+ T cells both in early and late phases, we observed the induction of antigen-specific TRM cells in the skin. (4) Conclusions: The OVA-EP-galDC vaccine efficiently induced antigen-specific antitumor immunity, which was sustained over time, as shown by the TRM cells.

Expansion of invariant natural killer T cells from systemic lupus erythematosus patients by alpha-Galactosylceramide and IL-15

CD1d-restricted invariant natural killer T cells (iNKT cells) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-15 is a pro-inflammatory cytokine which is over-expressed in SLE patients. In the present study, we investigated the iNKT cell expansion of mononuclear cells (MNCs) from SLE patients following 10 days’ culture with α-galactosylceramide (α-Galcer) and /or IL-15. We sought to determine the phenotypic and functional characteristics of the expanded iNKT cells compared to healthy controls and correlated with disease activity. We observed that 1. The percentages of Vα24+/Vβ11+ iNKT cells following 10-day incubation was lower in SLE groups compared to controls; 2. The percentages and absolute numbers of Vα24+/Vβ11+ iNKT cells were expanded by α-galactosylceramide (α-Galcer), and further enhanced with IL-15 in SLE patient, but the effect of IL-15 was much lower than controls; 3.IL-15 +α-Galcer expanded CD3+/CD56+ NKT-like cells from SLE patients, especially with active disease 4. The CD161+ Vα24+/Vβ11+ iNKT cells in SLE were more responsive to α-Galcer stimulation than the CD161- counterpart; 5. IL-15 decreased apoptosis of α-Galcer activated SLE iNKT cells; 6. IL-15 enhanced CD69, CD1d and CD11a expression on α-Galcer treated iNKT cells; 7. The IL-4 production of iNKT cells was decreased in SLE patients compared to controls; 8. IL-15 increased IFN-γ and IL-4 production of SLE iNKT cells; 8. IL-15 failed to augment the ability of iNKT cells to aid NK-mediated K562 cytolysis in SLE patients; 9. CD161 positivity, granzyme B and perforin expression of α-Galcer+IL-15 expanded iNKT cells correlated with C3 levels in SLE patients. Taken together, our results demonstrated numeric and functional deficiency of iNKT cells and their response to IL-15 in SLE patients. Our finding may provide insight for using adoptive iNKT cell therapy in autoimmune diseases.

Migration, Distribution, and Safety Evaluation of Specific Phenotypic and Functional Mouse Spleen-Derived Invariant Natural Killer T2 Cells after Adoptive Infusion

Herein, the migration distribution and safety of specific phenotypic and functionally identified spleen-derived invariant natural killer T2 (iNKT2) cells after adoptive infusion in mice were studied. The proliferation and differentiation of iNKT cells were induced by intraperitoneal injection of α-galactosylceramide (α-GalCer) in vivo. Mouse spleens were isolated in a sterile environment. iNKT cells were isolated by magnetic-activated cell sorting columns (MS columns). Cytometric bead array (CBA) assay was used to detect cytokine secretion in the supernatant stimulated by iNKT cells. The basic life status of the mice was observed, and systematic quantitative scoring was conducted after injecting spleen-derived iNKT cells through the tail vein. An in vivo imaging system was used to trace the migration and distribution of iNKT cells in DBA mice. The percentage of the iNKT2 subgroup was the highest in 3 days after intraperitoneal injection of α-GalCer, and iNKT2 subsets accounted for more than 92% after separation and purification by magnetic-activated cell sorting (MACS). Anti-inflammatory cytokine IL-4 was mainly found in the supernatant of cell cultures. The adoptive infusion of iNKT cells into healthy mice resulted in no significant change in the basic life status of mice compared with the noninjected group. iNKT cells were detected in the lung, spleen, and liver, but no fluorescence was detected in lymph nodes and thymus. After dissecting the mice, it was found that there were no significant abnormalities in the relevant immune organs, brain, heart, kidney, lung, and other organs. Intraperitoneal injection of α-GalCer results in a large number of iNKT2 cells, mainly secreting anti-inflammatory cytokine IL-4, from the spleen of mice. After adoptive infusion, the iNKT2 cells mainly settled in the liver and spleen of mice with a satisfactory safety profile.

The Role of Langerin⁺ CD8α⁺ Dendritic Cells in Tumour Immunotherapy

<p>The immune system has the potential to selectively target and eliminate tumours cells. However, the induction of an immunosuppressive environment by factors released by tumours cells, or by the tumour stroma, in combination with difficulties in differentiating between healthy and malignant cells, contributes to inefficient or disabled anti-tumour immune responses. A variety of different immunotherapeutic approaches are being developed to tip the balance in favour of anti-tumour immunity. Many of these approaches are designed to stimulate improved activity of T cells with specificity for tumour-associated antigens. This thesis explores how T cell-mediated responses are initiated and maintained in immunotherapy, with an emphasis on the role of antigen presentation by resident dendritic cells (DCs). An animal model was used in which a DC subset in the spleen that expresses the cell marker langerin could be selectively ablated during the course of therapy. As these DCs have been shown to be uniquely capable of acquiring circulating antigens and cellular debris, and have a heightened capacity for cross-priming CD8⁺ T cells, it was hypothesised that the function of these cells could play a significant role in determining the outcome of immunotherapies. A model of adoptive T cell therapy was examined in mice challenged with an intravenously administered lymphoma that formed tumour foci in a variety of locations in the body. Treating established tumours by adoptively transferring in vitro activated effector CD8⁺ T cells significantly increased their symptom-free survival. The protection received by this therapy was dependent on a stimulus being provided by endogenous langerin⁺ CD8α⁺ DCs to the transferred T cells. In the absence of langerin⁺ CD8α⁺ DCs, the proportion and number of transferred anti-tumour CD8⁺ T cells was lower in the blood and spleen. However, no obvious differences in phenotype and function could be defined. Langerin⁺ CD8α⁺ DCs therefore contribute to the maintenance of an effective CD8⁺ T cell-based immunotherapy and the role of endogenous DCs should be taken into consideration during the design of immunotherapies. To investigate the role of langerin⁺ CD8α⁺ DCs in initiating effector T cell responses, a novel whole-cell vaccine was developed for the treatment of acute myeloid leukaemia (AML). This vaccine exploited the stimulatory functions of invariant natural killer T cells, and was therefore administered intravenously to access the large invariant natural killer T cell compartment of the spleen. The vaccine completely protected mice from developing leukaemia when challenged with AML cells after vaccination, with CD4⁺ and CD8⁺ T cells mediating protection. The immune response generated by the vaccine was shown to be completely dependent on langerin⁺ CD8α⁺ DCs. In hosts with established tumours; however, the vaccine was ineffective. This may have been partially due to a reduced function of langerin⁺ CD8α⁺ DCs as their activation phenotype was significantly reduced in the presence of established AML; however, non-specific T cells could still be stimulated via these DCs. Reduced vaccine efficacy was associated with increased number and/or function of suppressor cells, including regulatory T cells and myeloid derived suppressor cells within the host. In addition, in leukemic hosts, the proportion of T cells in the spleen was reduced, and the function of AML-specific CD4⁺ T cells, but not CD8⁺ T cells, was impaired. Driving AML-bearing hosts into remission with chemotherapy prior to vaccination enabled the vaccine to protect the host from subsequent AML challenge. Langerin⁺ CD8α⁺ DCs are therefore responsible for initiating the vaccine-induced immune response in this model and their suppression may have contributed to the inefficacy of the vaccine in the presence of established tumours.</p>

The Role of Langerin⁺ CD8α⁺ Dendritic Cells in Tumour Immunotherapy

<p>The immune system has the potential to selectively target and eliminate tumours cells. However, the induction of an immunosuppressive environment by factors released by tumours cells, or by the tumour stroma, in combination with difficulties in differentiating between healthy and malignant cells, contributes to inefficient or disabled anti-tumour immune responses. A variety of different immunotherapeutic approaches are being developed to tip the balance in favour of anti-tumour immunity. Many of these approaches are designed to stimulate improved activity of T cells with specificity for tumour-associated antigens. This thesis explores how T cell-mediated responses are initiated and maintained in immunotherapy, with an emphasis on the role of antigen presentation by resident dendritic cells (DCs). An animal model was used in which a DC subset in the spleen that expresses the cell marker langerin could be selectively ablated during the course of therapy. As these DCs have been shown to be uniquely capable of acquiring circulating antigens and cellular debris, and have a heightened capacity for cross-priming CD8⁺ T cells, it was hypothesised that the function of these cells could play a significant role in determining the outcome of immunotherapies. A model of adoptive T cell therapy was examined in mice challenged with an intravenously administered lymphoma that formed tumour foci in a variety of locations in the body. Treating established tumours by adoptively transferring in vitro activated effector CD8⁺ T cells significantly increased their symptom-free survival. The protection received by this therapy was dependent on a stimulus being provided by endogenous langerin⁺ CD8α⁺ DCs to the transferred T cells. In the absence of langerin⁺ CD8α⁺ DCs, the proportion and number of transferred anti-tumour CD8⁺ T cells was lower in the blood and spleen. However, no obvious differences in phenotype and function could be defined. Langerin⁺ CD8α⁺ DCs therefore contribute to the maintenance of an effective CD8⁺ T cell-based immunotherapy and the role of endogenous DCs should be taken into consideration during the design of immunotherapies. To investigate the role of langerin⁺ CD8α⁺ DCs in initiating effector T cell responses, a novel whole-cell vaccine was developed for the treatment of acute myeloid leukaemia (AML). This vaccine exploited the stimulatory functions of invariant natural killer T cells, and was therefore administered intravenously to access the large invariant natural killer T cell compartment of the spleen. The vaccine completely protected mice from developing leukaemia when challenged with AML cells after vaccination, with CD4⁺ and CD8⁺ T cells mediating protection. The immune response generated by the vaccine was shown to be completely dependent on langerin⁺ CD8α⁺ DCs. In hosts with established tumours; however, the vaccine was ineffective. This may have been partially due to a reduced function of langerin⁺ CD8α⁺ DCs as their activation phenotype was significantly reduced in the presence of established AML; however, non-specific T cells could still be stimulated via these DCs. Reduced vaccine efficacy was associated with increased number and/or function of suppressor cells, including regulatory T cells and myeloid derived suppressor cells within the host. In addition, in leukemic hosts, the proportion of T cells in the spleen was reduced, and the function of AML-specific CD4⁺ T cells, but not CD8⁺ T cells, was impaired. Driving AML-bearing hosts into remission with chemotherapy prior to vaccination enabled the vaccine to protect the host from subsequent AML challenge. Langerin⁺ CD8α⁺ DCs are therefore responsible for initiating the vaccine-induced immune response in this model and their suppression may have contributed to the inefficacy of the vaccine in the presence of established tumours.</p>

iNKT subsets differ in their developmental and functional requirements on Foxo1

Invariant natural killer T (iNKT) cells play important roles in regulating immune responses. Based on cytokine profiling and key transcriptional factors, iNKT cells are classified into iNKT1, iNKT2, and iNKT17 subsets. However, whether the development and functions of these subsets are controlled by distinct mechanisms remains unclear. Here, we show that forkhead box protein O1 (Foxo1) promotes differentiation of iNKT1 and iNKT2 cells but not iNKT17 cells because of its distinct contributions to IL7R expression in these subsets. Nuclear Foxo1 is essential for Il7r expression in iNKT1 and iNKT2 cells at early stages of differentiation but is dispensable in iNKT17 cells. RORγt, instead of Foxo1, promotes IL7R expression in iNKT17 cells. Additionally, Foxo1 is required for the effector function of iNKT1 and iNKT2 cells but not iNKT17 cells. Cytoplasmic Foxo1 promotes activation of mTORC1 in iNKT1 and iNKT2 cells through inhibiting TSC1–TSC2 interaction, whereas it is dispensable for mTORC1 activation in iNKT17 cells. iNKT17 cells display distinct metabolic gene expression patterns from iNKT1 and iNKT2 cells that match their different functional requirements on Foxo1. Together, our results demonstrate that iNKT cell subsets differ in their developmental and functional requirements on Foxo1.

Ets1 Promotes the Differentiation of Post-Selected iNKT Cells through Regulation of the Expression of Vα14Jα18 T Cell Receptor and PLZF

The transcription factor Ets1 is essential for the development/differentiation of invariant Natural Killer T (iNKT) cells at multiple stages. However, its mechanisms of action and target genes in iNKT cells are still elusive. Here, we show that Ets1 is required for the optimal expression of the Vα14Jα18 T cell receptor (TCR) in post-selected thymic iNKT cells and their immediate differentiation. Ets1 is also critical for maintaining the peripheral homeostasis of iNKT cells, which is a role independent of the expression of the Vα14Jα18 TCR. Genome-wide transcriptomic analyses of post-selected iNKT cells further reveal that Ets1 controls leukocytes activation, proliferation differentiation, and leukocyte-mediated immunity. In addition, Ets1 regulates the expression of ICOS and PLZF in iNKT cells. More importantly, restoring the expression of PLZF and the Vα14Jα18 TCR partially rescues the differentiation of iNKT cells in the absence of Ets1. Taken together, our results establish a detailed molecular picture of how Ets1 regulates the stepwise differentiation of iNKT cells.

Engineering Human Invariant Natural Killer T (iNKT) Cells to Overexpress Immunomodulatory Cytokines

Many studies have shown the important role that invariant natural killer T (iNKT) cells play in suppression of graft-versus-host disease (GVHD). Murine studies have also demonstrated multiple subsets of iNKT cells, including those with pro-inflammatory and immunosuppressive properties. The prevention of murine acute GVHD by the adoptive transfer of immunomodulatory iNKT cells suggests that this approach would be ideal for clinical translation. However, the heterogeneity of human iNKT cells remains poorly understood, and the uncertain ability to identify immunosuppressive human iNKT cells and expand them to clinically relevant numbers remains a limitation in clinical translation. Therefore, engineering bulk human iNKT cells to enhance immunosuppressive capacity represents a novel approach to ensuring sufficient cell numbers of immunoregulatory cells for adoptive transfer. Herein, we describe the lentiviral transduction of human iNKT cells to overexpress immunomodulatory cytokines. We focused on IL-4, because murine iNKT cell-mediated GVHD suppression is IL-4 dependent, and IL-10, because an immunosuppressive regulatory T cell-like subset of murine iNKT cells highly produces IL-10. Human iNKT cells were transduced with a lentiviral construct expressing IL-4 or IL-10 (or green fluorescent protein (GFP) as a control) and an inert truncated nerve growth factor receptor (deltaNGFR) to facilitate sorting of transduced cells. Enzyme linked immunosorbent assay revealed significantly increased expression of IL-4 or IL-10 by cells transduced with the corresponding lentivirus, as compared to untransduced, mock transduced, or GFP transduced cells, replicated with 3 iNKT donors (p&lt;0.03) (Figure 1). This proof-of-concept data demonstrates the ability to effectively transduce human iNKT cells to overexpress immunomodulatory cytokines. Future studies will focus on evaluating the immunosuppressive capacity of these engineered cells both in vitro and in vivo (using the xenograft GVHD model). Successful suppression of GVHD by engineered iNKT cells will provide a critical step forward in identifying novel approaches to bring these promising cells to the clinic. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.