Transcription Factor T-bet Research Articles

Metabolic programs drive function of therapeutic NK cells in hypoxic tumor environments.

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rβ when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

The divergent outcome of IL-4Rα signalling on Foxp3 T regulatory cells in listeriosis and tuberculosis.

Forkhead box P3 (Foxp3) T regulatory cells are critical for maintaining self-tolerance, immune homeostasis, and regulating the immune system. We investigated interleukin-4 receptor alpha (IL-4Rα) signalling on T regulatory cells (Tregs) during Listeria monocytogenes (L. monocytogenes) infection using a mouse model on a BALB/c background, specifically with IL-4Rα knockdown in Tregs (Foxp3creIL-4Rα-/lox). We showed an impairment of Treg responses, along with a decreased bacterial burden and diminished tissue pathology in the liver and spleen, which translated into better survival. Mechanistically, we observed an enhancement of the Th1 signature, characterised by increased expression of the T-bet transcription factor and a greater number of effector T cells producing IFN-γ, IL-2 following ex-vivo stimulation with heat-killed L. monocytogenes in Foxp3creIL-4Rα-/lox mice. Furthermore, CD8 T cells from Foxp3creIL-4Rα-/lox mice displayed increased cytotoxicity (Granzyme-B) with higher proliferation capacity (Ki-67), better survival (Bcl-2) with concomitant reduced apoptosis (activated caspase 3). In contrast to L. monocytogenes, Foxp3creIL-4Rα-/lox mice displayed similar bacterial burdens, lung pathology and survival during Mycobacterium tuberculosis (M. tuberculosis) infection, despite increased T cell numbers and IFN-γ, TNF and IL-17 production. Our results demonstrated that the diminished IL-4Rα signalling on Foxp3+ T regulatory cells resulted in a loss of their functionality, leading to survival benefits in listeriosis but not in tuberculosis.

PP2A negatively regulates NK cell T-bet expression and anti-tumor effector function.

The transcription factor T-bet is essential for the anti-tumor effector function of NK cells, but the mechanism regulating its expression in NK cells remains unclear. In this study, we aimed to identify an NK cell intrinsic regulator that controls T-bet expression. Using T-bet-luciferase reporter assay screening, we identified a protein phosphatase inhibitor as a potential activator of T-bet expression. A series of PP2A-specific inhibitors (PP2Ai) or PP2A siRNA induced the expression of T-bet. In PP2Ai-treated mice, the expressions of T-bet and its downstream effector molecules, granzyme B and IFN-γ, were also upregulated in NK cells. Mechanistically, PP2Ai increased the phosphorylation of mTOR and ribosomal protein S6 in NK cells, and mTOR inhibitor canceled the effects of PP2Ai in NK cells. Importantly, NK cells isolated from PP2Ai-treated mice showed higher cytotoxicity and IFN-γ production; therefore, they increased the anti-tumor effector function of NK cells. Accordingly, PP2Ai treatment inhibited lung metastasis of B16 melanoma by NK cell- and mTOR-dependent mechanisms. These results suggest that PP2A negatively regulates NK cell T-bet expression and effector function by an mTOR-dependent mechanism.

450. TISSUE RESIDENT MEMORY CELLS: CORRELATING THE PHENOTYPIC AND FUNCTIONAL CAPABILITIES OF A POTENTIAL TUMOUR REACTIVE CELL POPULATION IN OESOPHAGEAL ADENOCARCINOMA

Abstract Background Oesophageal adenocarcinoma (OAC) is a disease with a high mortality and morbidity rate. Immunotherapy has recently demonstrated progress in the treatment of OAC but its impact on long term survival is currently uncertain. Tissue resident memory (TRM) T cells are a subset of lymphocytes within the tumour/tissue infiltrating lymphocyte (TIL) milieu that has attracted considerable interest within cancer immunology. Although TRM cells have been associated with better outcomes in multiple cancer types there is a lack of knowledge of their role in OAC. This study aims to critically correlate the phenotype of TRM with their functional capacity in this disease. Methods 33 patients undergoing surgical resection for OAC were recruited and consented for collection of fresh tumour (T), adjacent normal tissue (AN), and peripheral blood. Peripheral blood mononuclear cell (PBMC) from blood and infiltrating lymphocytes from T and AN were isolated using standard density gradient and enzymatic digestion protocols. Multiparametric flow cytometry was performed on 12 patients using matched PBMC, T and AN infiltrating lymphocytes for phenotypic analysis and 21 patients for functional analysis following cell stimulation assay. BD FACSymphony was used for data acquisition and data analysed on FlowJo version 10.10. Statistical analysis was carried out using GraphPad Prism. Results TRM (CD103+CD69+) dominated the CD8+ T cell population of T and AN TIL. Expression of checkpoint proteins PD1+ and CD39+, a marker of tumour reactivity, was identified on CD8+ TRM and CD8+ non TRM in TIL. The differentiation status of TRM within memory lineages demonstrated that TRM express a higher proportion of effector memory cells (CCR7-CD45RA) compared to those in blood. TRM cells also showed lower expression of the transcription factors EOMES, TBET and TCF compared to CD8+ cells within PBMC. Moreover, tumour CD8+ TRM populations had reduced ability to produce IL-2 following stimulation compared to adjacent normal TRM populations. Conclusion TRM make up a large proportion of CD8+ T cell within the tumour microenvironment (TME) of OAC. TRM generation is associated with the downregulation of T-Box transcription factors EOMES and TBET and expression of an effector memory phenotype with reduction of the stem-like transcription factor, TCF. High levels of CD39+ indicate a strong level of activation but the increased PD1 expression and reduced capability to produce IL2 indicate functional impairment suppressed within the TME. An opportunity may exist to reverse this with novel immunotherapeutic agents.

An intestinal TH17 cell-derived subset can initiate cancer

Approximately 25% of cancers are preceded by chronic inflammation that occurs at the site of tumor development. However, whether this multifactorial oncogenic process, which commonly occurs in the intestines, can be initiated by a specific immune cell population is unclear. Here, we show that an intestinal T cell subset, derived from interleukin-17 (IL-17)-producing helper T (TH17) cells, induces the spontaneous transformation of the intestinal epithelium. This subset produces inflammatory cytokines, and its tumorigenic potential is not dependent on IL-17 production but on the transcription factors KLF6 and T-BET and interferon-γ. The development of this cell type is inhibited by transforming growth factor-β1 (TGFβ1) produced by intestinal epithelial cells. TGFβ signaling acts on the pretumorigenic TH17 cell subset, preventing its progression to the tumorigenic stage by inhibiting KLF6-dependent T-BET expression. This study therefore identifies an intestinal T cell subset initiating cancer.

Interconnected lineage trajectories link conventional and natural killer (NK)-like exhausted CD8+ T cells beneficial in type 1 diabetes

Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.

Plasticity and lineage commitment of individual TH1 cells are determined by stable T-bet expression quantities.

T helper 1 (TH1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated TH1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus-induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the TH2 lineage: T-bet quantities were inversely correlated with the ability to express the TH2 lineage-specifying transcription factor GATA-3 and TH2 cytokines. Reprogramed TH1 cells acquired graded mixed TH1 + TH2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated TH1 cells was essential to ensure TH1 cell stability. Thus, innate cytokine signals regulate TH1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.

FOXP3+ Regulatory T Cells Require TBET to Regulate Activated CD8+ T Cells During Recovery from Influenza Infection.

FOXP3+ regulatory T (Treg) cells are necessary to coordinate resolution of lung inflammation and a return to homeostasis after respiratory viral infections, but the specific molecular requirements for these functions and the cell types governed by Treg cells remain unclear. This question holds significance as clinical trials of Treg cell transfer therapy for respiratory viral infection are being planned and executed. Here, we report causal experiments in mice determining that Treg cells are necessary to control the numbers of activated CD8+ T cells during recovery from influenza infection. Using a genetic strategy paired with adoptive transfer techniques, we determined that Treg cells require the transcription factor TBET to regulate these potentially pro-inflammatory CD8+ T cells. Surprisingly, we found that Treg cells are dispensable for the generation of CD8+ lung tissue resident-memory T (Trm) cells yet similarly influence the transcriptional programming of CD8+ Trm and activated T cells. Our study highlights the role of Treg cells in regulating the CD8+ T cell response during recovery from influenza infection.

Effect of ADSCs on Th17/Treg and T-bet/GATA-3 in model mice with primary immune thrombocytopenia.

We aimed to observe the effects of adipose-derived mesenchymal stem cells (ADSCs) on T helper 17 (Th17)/regulatory T cells (Treg) and T-box transcription factor (T-bet)/GATA-binding protein 3 (GATA-3) in model mice with primary immune thrombocytopenia (ITP). 32 BALB/C mice were selected. ADSCs were isolated from 2 mice and cultured. The other 30 mice were randomly divided into the normal control group, the ITP model control group, and the ITP experimental group. Platelet count (PLT), Th17/Treg cells, related serum cytokines [interleukin-6 (IL-6), IL-17A, IL-10, and transforming growth factor β1 (TGF-β1)], T-bet and GATA-3 mRNA levels in peripheral blood mononuclear cells (PBMCs) in the 3 groups were detected. PLT and Treg in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). Th17 and Th17/Treg in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-6 and IL-17A levels, and T-bet mRNA levels in the ITP experimental group were significantly higher than those in the normal control group (P<0.05), but significantly lower than those in the ITP model control group (P<0.05). Serum IL-10 and TGF-β levels, and GATA-3 mRNA levels in the ITP experimental group were significantly lower than those in the normal control group (P<0.05), but significantly higher than those in the ITP model control group (P<0.05). ADSCs can effectively regulate Th17/Treg balance and improve T-bet/GATA-3 mRNA expression levels in ITP model mice.

Acupuncture at "Die E acupoint" alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis.

To observe effects of acupuncture at "Die E acupoint" on the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB), transcription factor T-bet (T-bet), and GATA-binding protein-3 (GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis, so as to explore the mechanism of acupuncture in treating allergic rhinitis. Twenty-four healthy SD rats were randomly divided into blank, model, acupuncture, and sham acupuncture groups, with 6 rats in each group. The rat model of allergic rhinitis was established by using ovalbumin induction. The rats in the acupuncture group received bilateral acupuncture at the "Die E acupoint" with a depth of 15-20 mm, while the rats in the sham acupuncture group received only sham acupuncture (light and shallow acupunture of the skin at the "Die E acupoint" ). Both interventions were performed once daily for a total of 6 days. Behavioral scores of rats in each group were recorded. Pathological changes of nasal mucosa were observed by H.E. staining. Serum contents of IgE, ovalbumin-specific IgE (OVA-sIgE), interferon(IFN)-γ, interleukin(IL)-4, IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet, GATA-3, TLR4, MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot. After modeling, compared with the blank group, rats in the model group showed increased behavioral scores, serum IgE, OVA-sIgE, IL-4, and IL-17 contents, and nasal mucosal GATA-3, TLR4, MyD88, and NF-κB p65 protein expression levels (P<0.05), whereas the contents of serum IFN-γ, IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased (P<0.05). Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis, the contents of serum IgE, OVA-sIgE, IL-4, IL-17, and the protein expression levels of GATA-3, TLR4, MyD88, and NF-κB p65 in the nasal mucosa (P<0.05), and up-regulate the contents of serum IFN-γ, IL-10, and the nasal mucosal T-bet protein expression level. Sham acupuncture did not have a significant modulating effect on the above indicators. Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture, and the inflammatory reaction was milder in the acupuncture group. Acupuncture at "Die E acupoint" can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats, which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17, and T-bet/GATA-3.

Cang-ai volatile oil alleviates nasal inflammation via Th1/Th2 cell imbalance regulation in a rat model of ovalbumin-induced allergic rhinitis.

We previously revealed that Cang-ai volatile oil (CAVO) regulates T-cell activity, enhancing the immune response in people with chronic respiratory diseases. However, the effects of CAVO on allergic rhinitis (AR) have not been investigated. Herein, we established an ovalbumin (OVA)-induced AR rat model to determine these effects. Sprague-Dawley (SD) rats were exposed to OVA for 3weeks. CAVO or loratadine (positive control) was given orally once daily for 2weeks to OVA-exposed rats. Behavior modeling nasal allergies was observed. Nasal mucosa, serum, and spleen samples of AR rats were analyzed. CAVO treatment significantly reduced the number of nose rubs and sneezes, and ameliorated several hallmarks of nasal mucosa tissue remodeling: inflammation, eosinophilic infiltration, goblet cell metaplasia, and mast cell hyperplasia. CAVO administration markedly upregulated expressions of interferon-γ, interleukin (IL)-2, and IL-12, and downregulated expressions of serum tumor necrosis factor-α, IL-4, IL-5, IL-6, IL-13, immunoglobulin-E, and histamine. CAVO therapy also increased production of IFN-γ and T-helper type 1 (Th1)-specific T-box transcription factor (T-bet) of the cluster of differentiation-4+ T-cells in splenic lymphocytes, and protein and mRNA expressions of T-bet in nasal mucosa. In contrast, levels of the Th2 cytokine IL-4 and Th2-specific transcription factor GATA binding protein-3 were suppressed by CAVO. These cumulative findings demonstrate that CAVO therapy can alleviate AR by regulating the balance between Th1 and Th2 cells.

Th1/Th2 associated transcription factors and cytokines in pregnancies with systemic lupus erythematosus.

Th1/Th2 shift occurs during pregnancy. Systemic lupus erythematosus (SLE) flares may induce the dysregulation of Th1 and Th2 cells. We aimed to investigate the dynamic changes of Th1/Th2 associated transcription factors and cytokines in patients with SLE during pregnancy. Twenty-five pregnant patients with SLE and twenty-two healthy age-matched women served as controls from September 2021 to March 2022 were enrolled in the study. Real-time quantitative reverse transcription polymerase chain reaction analysis of peripheral blood mononuclear cells were performed to measure the expression of Th1 specific transcription factors T-bet, cytokine IFN-γ, and Th2 specific transcription factors GATA3, cytokine IL-4. The main statistical analysis methods were t test, Mann-Whitney U-test, Pearson correlation and Spearman rank correlation analysis. The mRNA level of IFN-γ and the relative expression of T-bet/GATA3 and IFN-γ/IL-4 in SLE patients were significantly higher than those in healthy individuals, whereas the GATA3 expression is lower in pregnant patients with SLE (p<0.001, p<0.05, p<0.05 and p<0.01 during the whole pregnancy, respectively; p<0.05, p<0.01, p<0.05 and p<0.05 specifically for the 3rd trimester, respectively). There were significant correlations between T-bet and IFN-γ (r=0.492, p<0.05), and between T-bet/GATA3 and IFN-γ/IL-4 (r=0.482, p<0.05). Our work indicates that in SLE patients Th1/Th2 shift is blocked with up-regulation of Th1 cell function and insufficient Th2 polarisation during pregnancy, which may be involved in adverse pregnancy outcomes.

T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia

AbstractThe T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.

Targeting T-bet expressing B cells for therapeutic interventions in autoimmunity.

Apart from serving as a Th1 lineage commitment regulator, transcription factor T-bet is also expressed in other immune cell types and thus orchestrates their functions. In case of B cells, more specifically, T-bet is responsible for their isotype switching to specific IgG sub-classes (IgG2a/c in mice and IgG1/3 in humans). In various autoimmune disorders, such as systemic lupus erythematosus and/or rheumatoid arthritis, subsets of T-bet expressing B cells, known as age-associated B cells (CD19+CD11c+CD21-T-bet+) and/or double-negative B cells (CD19+IgD-CD27-T-bet+), display an expansion and seem to drive disease pathogenesis. According to data, mostly derived from mice models of autoimmunity, the targeting of these specific B-cell populations is capable of ameliorating the general health status of the autoimmune subjects. Here, in this review article, we present a variety of therapeutic approaches for both mice and humans, suffering from an autoimmune disease, and we discuss the effects of each approach on T-bet+ B cells. In general, we highlight the importance of specifically targeting T-bet+ B cells for therapeutic interventions in autoimmunity.

Enrichment of type 1 innate lymphoid cells in the course of human atherosclerotic plaque development suggests contribution to atherogenesis.

Innate lymphoid cells (ILCs) have been implicated in multiple pathologic conditions, including atherogenesis, as documented in experimental mice studies, however, their role in atherosclerosis in humans remains unexplored. Here, we identify ILCs and their dynamics in early, advanced, and complicated human carotid- and aortic atherosclerotic plaques, using a multiplex immunohistochemical quadruple-staining technique with prototypic transcription factors T-bet, GATA3, or RORgt for identification of the ILC1, ILC2 and ILC3 subsets, respectively, in combination with lineage markers CD3, CD20/ CD79a and CD56 to exclude other lymphoid cell types. ILC subsets were quantified, and to put this in perspective, their numbers were expressed as percentage of the total number of infiltrated lymphoid cells and related to the frequency of conventional T cells, B cells, NK cells, and NKT cells. All ILC subsets were present in every different stage of atherogenesis. ILC1s were the most abundant ILC subset, and their numbers significantly increased in the course of plaque development, but paradoxically, their relative frequency was reduced because of a higher increment of T cells and B cells. The numbers of ILC2s and ILC3s also gradually increased, but this trend did not achieve significance. T cell subsets always significantly outnumbered their ILC counterparts, except for the early lesions where the proportion of ILC1s was markedly higher, albeit not significant. The high abundance of ILC1s in the early stages and further significant enrichment in later stages, suggest they may participate in the initiation and development of atherogenesis, and thus, may represent a novel target to prevent or treat atherosclerosis.

Group 1 ILCs: Heterogeneity, plasticity, and transcriptional regulation.

Group 1 innate lymphoid cells (ILCs), comprising ILC1s and natural killer cells (NK cells), belong to a large family of developmentally related innate lymphoid cells that lack rearranged antigen-specific receptors. NK cells and ILC1s both require the transcription factor T-bet for lineage commitment but additionally rely on Eomes and Hobit, respectively, for their development and effector maturation programs. Both ILC1s and NK cells are essential for rapid responses against infections and mediate cancer immunity through production of effector cytokines and cytotoxicity mediators. ILC1s are enriched in tissues and hence generally considered tissue resident cells whereas NK cells are often considered circulatory. Despite being deemed different cell types, ILC1s and NK cells share many common features both phenotypically and functionally. Recent studies employing single cell RNA sequencing (scRNA-seq) technology have exposed previously unappreciated heterogeneity in group 1 ILCs and further broaden our understanding of these cells. Findings from these studies imply that ILC1s in different tissues and organs share a common signature but exhibit some unique characteristics, possibly stemming from tissue imprinting. Also, data from recent fate mapping studies employing Hobit, RORγt, and polychromic reporter mice have greatly advanced our understanding of the developmental and effector maturation programs of these cells. In this review, we aim to outline the fundamental traits of mouse group 1 ILCs and explore recent discoveries related to their developmental programs, phenotypic heterogeneity, plasticity, and transcriptional regulation.

Abstract 2685: Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers

Abstract Innate lymphoid cells (ILCs) and natural killer (NK) cells are a heterogeneous family of innate immune cells that regulate inflammation, immune tolerance and tissue homeostasis in infection, chronic inflammation, metabolic diseases and cancer. The ILC family lacks the expression of antigen-specific receptors and demonstrate effector function by generating cytokines and secreted proteins. In tumor microenvironment, ILCs play a dual role including both tumor regression and tumor promotion, suggesting their diverse phenotypes and functional states. Among the ILC family, ILC1s and NK cells share some features, including the expression of the transcription factor T-bet and the ability to produce IFN-γ. In the present study, we analyzed single-cell RNA sequencing (scRNAseq) data to investigate the landscape and functional status of ILC families in patients with head and neck squamous cell carcinoma (HNSCC). The GSE164690 dataset, including pre-processed scRNAseq data and clinical data, was obtained from Gene Expression Omnibus. A total of 17 pairs of tumor-derived CD45-positive cells and peripheral blood lymphocytes, consisting of 6 HPV-positive HNSCCs and 11 HPV-negative HNSCCs, were analyzed using the Seurat v4 R package. After quality control, a total of 95,809 cells were obtained and clustered into 16 immune cell clusters. Next, a total of 7,278 NK cells were extracted and sub-clustered into 11 clusters. To characterize the 11 clusters, the abundances of NK subsets were estimated by CIBERSORTx. This approach identified eight NK cell clusters, two intraepithelial ILC1 (ieILC1) clusters, and one ieILC1-NK-intermediate (ieILC1-NK-int) cluster. Single-sample GSEA (ssGSEA) analysis was performed to further characterize each subset. ieILC1 clusters expressed tissue-resident markers ITGA1, CD69, and CXCR6, whereas ieILC1-NK-int cluster expressed ILC1 markers CCR7, IL7R, and SELL. Principle component analysis (PCA) based on ssGSEA indicated that ieILC1 clusters and ieILC1-NK-int showed the different PCA distribution from NK clusters. Inflammation and effector function-related pathways, including inflammatory response, interferon-gamma response, and interferon-alpha response, were enriched in ieILC1 clusters compared with the others. Among ieILC1 clusters, several pathways, including hypoxia, TNFA signaling via NFKB, allograft rejection, TGF beta signaling, were more enriched in ieILC1-1 than in ieILC1-2, suggesting that ieILC1-1 is the most immunologically active phenotype among NK subclusters. Of note, ieILC1-2 mainly comprised cells isolated from HPV-negative tumors, whereas ieILC1-1 mainly comprised cells isolated from HPV-positive tumors. In conclusion, we have demonstrated the diverse landscape of NK and ieILC1 cells in patients with HNSCC. Further characterization of the ILC family would lead to a novel therapeutic approach against HNSCC. Citation Format: Hideyuki Takahashi, Shota Ida, Hiroe Tada, Kazuaki Chikamatsu. Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2685.

Abstract 3973: Cancer B cell blueprint reveals dysfunctional extra-follicular dominance with therapeutic opportunity

Abstract B cells are essential components of humoral immunity but harbor multifaceted functions across cancers. To decode tumor-infiltrating B cells, we generated the B cell blueprint encompassing single-cell transcriptome, BCR repertoire, and chromatin accessibility data across 20 cancer types (483 samples, 266 patients). Strikingly, B cells harbored extraordinary heterogeneity and composed of 15 subsets, which were grouped into two independent developmental paths (extra-follicular [EF] versus germinal-center [GC]) exhibiting organ preference. Of note, colon adenocarcinoma and hepatocellular carcinoma are the two representative types of cancer enriched for GC and EF pathways, respectively. We affirm that EF-dominant cancers correlate with dysregulated immune responses and worse clinical outcomes. We then identify the dynamic metabolic-epigenetic-signaling networks in fine-tuning tumor-infiltrating B cell differentiation and managing the balance between the EF and GC pathways. Atypical memory (AtM) B cells, the primary progenitors of EF-derived ASCs, exhibit an exhausted and bystander phenotype and develop independently of the GC pathway. We find that the AtM B cells reside in the center of immature TLSs and spatially relocate to the periphery during TLS maturation. Finally, we mechanistically link these findings to specific transcription factors and epigenomic regulations. We demonstrate that the glutamine-derived metabolite α-ketoglutarate (αKG) could increase the expression of AtM B cell associated transcription factors Tbet and BATF and promote their differentiation, accompanied by the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Consequently, AtM B cells acquire an immunoregulatory function that dampens anti-tumor T cell responses and fosters an immunosuppressive microenvironment. Citation Format: Jiaqiang Ma, Yingcheng Wu, Lifeng Ma, Xupeng Yang, Tiancheng Zhang, Guoji Guo, Jia Fan, Xiaoming Zhang, Qiang Gao. Cancer B cell blueprint reveals dysfunctional extra-follicular dominance with therapeutic opportunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3973.

Elotuzumab Enhances CD16-Independent NK Cell-Mediated Cytotoxicity against Myeloma Cells by Upregulating Several NK Cell-Enhancing Genes.

Multiple myeloma (MM) is an intractable hematological malignancy caused by abnormalities in plasma cells. Combination therapy using antibodies and natural killer (NK) effectors, which are innate immune cells with safe and potent antitumor activity, is a promising approach for cancer immunotherapy and can enhance antitumor effects. Elotuzumab (Elo) is an immune-stimulatory antibody that targets the signaling lymphocytic activation molecule family 7 (SLAMF7) expressed on the surface of MM and NK cells. We confirmed that Elo strongly promoted NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SLAMF7-positive MM cells in a CD16-dependent NK cell line, and also activated expanded NK cells derived from peripheral blood mononuclear cells of healthy donors and patients with MM in the present study. However, the antitumor effects and genes involved in the direct promotion of NK cell-mediated activation using Elo in CD16-independent NK cells are not clearly known. In this study, we demonstrated that Elo pretreatment significantly enhanced CD16-independent NK cell-mediated cytotoxicity in both SLAMF7-positive MM.1S and SLAMF7-negative K562, U266, and RPMI 8226 tumor cells. Upon direct simulation of CD16-independent NK cells with Elo, increased levels of CD107a degranulation and IFN-γ secretion were observed along with the upregulation of granzyme B, TNF-α, and IL-1α gene expression. The enhanced NK cell function could also be attributed to the increased expression of the transcription factors T-BET and EOMES. Furthermore, the augmentation of the antitumor effects of CD16-independent NK cells upon pretreatment with Elo enhanced the expression of CRTAM, TNFRSF9, EAT-2, and FOXP3 genes and reduced the expression of HSPA6. Our results suggest that Elo directly promotes the cytotoxic function of CD16-independent NK cells against target cells, which is associated with the upregulation of the expression of several NK cell-enhancing genes.

Two regulatory T cell populations in the visceral adipose tissue shape systemic metabolism.

Visceral adipose tissue (VAT) is an energy store and endocrine organ critical for metabolic homeostasis. Regulatory T (Treg) cells restrain inflammation to preserve VAT homeostasis and glucose tolerance. Here, we show that the VAT harbors two distinct Treg cell populations: prototypical serum stimulation 2-positive (ST2+) Treg cells that are enriched in males and a previously uncharacterized population of C-X-C motif chemokine receptor 3-positive (CXCR3+) Treg cells that are enriched in females. We show that the transcription factors GATA-binding protein 3 and peroxisome proliferator-activated receptor-γ, together with the cytokine interleukin-33, promote the differentiation of ST2+ VAT Treg cells but repress CXCR3+ Treg cells. Conversely, the differentiation of CXCR3+ Treg cells is mediated by the cytokine interferon-γ and the transcription factor T-bet, which also antagonize ST2+ Treg cells. Finally, we demonstrate that ST2+ Treg cells preserve glucose homeostasis, whereas CXCR3+ Treg cells restrain inflammation in lean VAT and prevent glucose intolerance under high-fat diet conditions. Overall, this study defines two molecularly and developmentally distinct VAT Treg cell types with unique context- and sex-specific functions.