The cytotoxicity feature to eliminate malignant cells makes natural killer (NK) cells a candidate for tumor immunotherapy. However, this scenario is currently hampered by inadequate understanding of the regulatory mechanisms of NK cell development. Ten-Eleven-Translocation 2 (Tet2) is a demethylase whose mutation was recently shown to cause phenotypic defects in NK cells. However, the role of Tet2 in the development and maturation of NK cells is not entirely clear. Here we studied the modulatory role of Tet2 in NK cell development and maturation by generating hematopoietic Tet2 knockout mice and mice with Tet2 conditional deletion in NKp46+ NK cells. The results showed that both hematopoietic and NK cell conditional deletion of Tet2 had no effect on the early steps of NK cell development, but impaired the terminal maturation of NK cells defined by CD11b, CD43, and KLRG1 expression. In the liver, Tet2 deletion not only prevented the terminal maturation of NK cells, but also increased the proportion of type 1 innate lymphoid cells (ILC1s) and reduced the proportion of conventional NK cells (cNK). Moreover, hematopoietic deletion of Tet2 lowered the protein levels of perforin in NK cells. Furthermore, hematopoietic deletion of Tet2 downregulated the protein levels of Eomesodermin (Eomes), but not T-bet, in NK cells. In conclusion, our results demonstrate that Tet2 plays an important role in the terminal maturation of NK cells, and the Eomes transcription factor may be involved.

Eomesodermin (Eomes) is a critical factor in the development of natural killer (NK) cells, but its precise role in temporal and spatial coordination during this process remains unclear. Our study revealed that Eomes plays distinct roles during the early and late stages of NK cell development. Specifically, the early deletion of Eomes via the CD122-Cre transgene resulted in significant blockade at the progenitor stage due to the downregulation of KLF2, another important transcription factor. ChIP-seq revealed direct binding of Eomes to the conserved noncoding sequence (CNS) of Klf2. Utilizing the CHimeric IMmune Editing (CHIME) technique, we found that deletion of the CNS region of Klf2 via CRISPRi led to a reduction in the NK cell population and developmental arrest. Moreover, constitutive activation of this specific CNS region through CRISPRa significantly reversed the severe defects in NK cell development caused by Eomes deficiency. Conversely, Ncr1-Cre-mediated terminal deletion of Eomes expedited the transition of NK cell subsets from the CD27+CD11b+ phenotype to the CD27-CD11b+ phenotype. Late-stage deficiency of Eomes led to a significant increase in T-bet expression, which subsequently increased the expression of the transcription factor Zeb2. Genetic deletion of one allele of Tbx21, encoding T-bet, effectively reversed the aberrant differentiation of Eomes-deficient NK cells. In summary, we utilized two innovative genetic models to elucidate the intricate mechanisms underlying Eomes-mediated NK cell commitment and differentiation.

The activation and specialization of regulatory T cells (Tregs) are crucial for maintaining immune self-tolerance; however, the regulation of these processes by histone modifications is not fully understood. Here, we show that T cell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional T cells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. Moreover, RNA sequencing demonstrates that the induction of multiple genes involved in Treg activation, functional specialization, and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at these genes\' transcription start sites. Finally, using a T-bet fate-mapping mouse system, we determine that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 is essential in optimal Treg function by providing a coordinated chromatin context for activation and specialization.

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.

Innate lymphoid cells (ILCs) are tissue-resident immune cells that have been recently implicated in initiating and driving anti-tumor responses. ILCs are classified into three main groups, namely type 1 ILCs (ILC1), type 2 ILCs, and type 3 ILCs. All three groups have been implicated in either eliciting pro or anti-tumor immune responses in different cancer subtypes with the consensus that ILCs cannot be overlooked within the field of anti-tumor immune responses. In this review, we will specifically expand on the knowledge on ILC1, their characterization, function, and plasticity in anti-cancer immune responses. Within this premise, we will discuss caveats of ILC1 characterization, and expand on the expression and function of immune checkpoint receptors within ILC1 subsets, specifically focusing on the role of programmed cell death-1 receptor in controlling specific ILC1 responses. We summarize that ILC1s are a vital component in initiating anti-tumor responses and can be boosted by checkpoint receptors.

The NK cell exhaustion state evolving during extensive and prolonged cultivation is still one of the limitations of NK cell approaches. In this research, we transduced NK cells with the hTERT and iCasp9 genes. hTERT overexpression can prevent the functional exhaustion of NK cells during long-term cultivation, but, still, the therapeutic use of such cells is unsafe without irradiation. To overcome this obstacle, we additionally transduced NK cells with the iCasp9 transgene that enables the rapid elimination of modified cells. We compared the proliferative and functional activities of the hTERT- and/or iCasp9-modified NK cells, determined their exhaustion state and monitored the levels of EOMES and T-BET, the main NK cell transcription factors. The hTERT and iCasp9 genes were shown to affect the EOMES and T-BET levels differently in the NK cells. The EOMES+T-BET+ phenotype characterized the functionally active NK cells during two months of culture upon stimulation with IL2 and K562-mbIL21 feeder cells, which induced the greatest expansion rates of the NK cells, independently of the transgene type. On the other hand, under cytokine stimulation, the hTERT-iCasp9-NK cells displayed improved proliferation over NK cells modified with iCasp9 alone and showed an increased proliferation rate compared to the untransduced NK cells under stimulation with IL2 and IL15, which was accompanied by reduced immune checkpoint molecule expression. The individual changes in the EOMES and T-BET levels strictly corresponded to the NK cell functional activity, the surface levels of activating and inhibitory receptors along with the expansion rate and expression levels of pro-survival and pro-apoptotic genes.

Myeloid-derived suppressor cells (MDSC) are a group of immature inhibitory cells of bone marrow origin. Human γδ T cells (mainly Vγ9Vδ2 T cells) have emerged as dominant candidates for cancer immunotherapy because of their unique recognition pattern and broad killing activity against tumor cells. Intestinal mucosal intraepithelial lymphocytes are almost exclusively γδ T cells, so it plays an important role in inhibiting the development of colorectal cancer. In this study, we investigated the effects and molecular mechanism of human MDSC on anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our results suggested that MDSC can reduce the NKG2D expression of Vγ9Vδ2 T cells through direct cell-cell contact, which is associated with membrane-type transforming growth factor-β. In contrast, MDSC can increase Vγ9Vδ2 T cells activation and production of IFN-γ, perforin, Granzyme B through direct cell-cell contact. This may be related to the upregulation of T-bet in Vγ9Vδ2 T cells by MDSC. However, MDSC had a dominant negative regulatory effect on the anticolorectal cancer cells activity of Vγ9Vδ2 T cells. Our study provides a theoretical basis for the immune regulatory function of human MDSC on γδ T cells. This will be conducive to the clinical development of a new antitumor therapy strategy.

Systemic lupus erythematosus (SLE) is an inflammatory disorder related to immunity dysfunction. The Th1 cell family including Th1 cells, transcription factor T-bet, and related cytokines IFNγ, TNFα, IL-2, IL-18, TGF-β, and IL-12 have been widely discussed in autoimmunity, such as SLE. In this review, we will comprehensively discuss the expression profile of the Th1 cell family in both SLE patients and animal models and clarify how the family members are involved in lupus development. Interestingly, T-bet-related age-associated B cells (ABCs) and low-dose IL-2 treatment in lupus were emergently discussed as well. Collection of the evidence will better understand the roles of the Th1 cell family in lupus pathogenesis, especially targeting IL-2 in lupus.

BACKGROUND: Evaluate the immunohistochemical expression of T-bet and IFN-γ in lower lip (LLSCC) and oral tongue squamous cell carcinoma (OTSCC), verifying the presence of Th1 responses in lesions with different clinical conditions.
METHODS: Thirty OTSCC and 30 LLSCC were analyzed by immunohistochemistry. T-bet was quantitatively assessed by parenchyma cell and stroma quantification, and IFN-γ was semi-quantitatively analyzed: 1:0-25%; 2:26-50%; 3:51-75%; 4:> 75% immunopositive cells. Histological differentiation degrees were categorized as well differentiated (WD), moderately differentiated (MD), or poorly differentiated (PD).
RESULTS: OTSCC presented the highest number of T-bet+, parenchyma (p: 0.006), stroma (p: 0.156), parenchyma/stroma (p: 0.015), with no relationship to histological malignancy grade. IFN-γ higher concentrations in LLSCC were detected in parenchyma, stroma and in parenchyma/stroma (p: 0.000), as well as greater immunoreactivity in WD and MD (p: 0.001). In OTSCC, a positive and statistically significant correlation was observed between T-bet+ in parenchyma and IFN-γ in stroma(r: 0.388; p: 0.034), in addition to a statistically significant positive correlation between T-bet in parenchyma compared to stroma(r: 0.411; p: 0.024) and for IFN-γ in both parenchyma and stroma(r: 0.775; p: 0.000) in LLSCC. Higher T-bet+ was observed in OTSCCs, although higher IFN-γ was detected in LLSCCs.
CONCLUSIONS: Thus, we suggest that, even though LLSCC presented lower T-bet+, the favorable microenvironment in these lesions led to an expressive activation of IFN-γ by T-bet+, considerably acting on Th1 differentiation and in antitumor activity, which, admittedly, present less aggressive behavior, reinforcing once again the important role of this cytokine and its use in strategy to fight cancer.

OBJECTIVE: The differentiation of CD4+ T cells is regulated by a complex and fine signaling pathway composed of many molecules during immune response, and the molecular mechanism for regulating T-bet expression is unclear. Mediator complex subunit 1 (Med1) can combine with a variety of co-factors to regulate gene transcription, promote cell proliferation and survival, and affect invariant natural killer T cell (iNKT) development. This study aims to investigate the effect of Med1 on T cell development and CD4+ T cell differentiation in immune response.
METHODS: Mice with T cell-specific knockout of Med1 gene (Med1F/FCD4cre+, KO) were constructed and verified. The percentage and number of CD4+ and CD8+ T cells in thymus, spleen, and lymph nodes of KO mice and control (Con) mice (Med1F/FCD4cre-) were detected by flow cytometry. After 8 days of infection with lymphocytic choriomeningitis virus (LCMV), the percentage and number of CD4+ T cells or antigen-specific (GP66+) CD4+ T cells, the percentage and number of Th1 cells (Ly6c+PSGL1+) in CD4+ T cells or antigen-specific CD4+ T cells were examined in the spleen of mice. Moreover, the fluorescence intensity of T-bet in CD4+ T cells or antigen-specific CD4+ T cells was analyzed.
RESULTS: Compared with the Con group, the percentage and number of CD4+ T cells and CD8+ T cells in the thymus, CD4+ T cells in the spleen and lymph nodes of the KO group showed no significant differences (all P>0.05), but the percentage and number of CD8+ T cells in the spleen and lymph nodes of the KO group were diminished significantly (all P<0.05). After 8 days of infection with LCMV, there was no significant difference in the percentage and number of CD4+ T cells or antigen-specific CD4+ T cells in the spleen between the KO group and the Con group (all P>0.05), while in comparison with the Con group, the percentage and number of Th1 cells in CD4+ T cells or antigen-specific CD4+ T cells, and the expression of T-bet in CD4+ T cells or antigen-specific CD4+ T cells were significantly reduced in the spleen of the KO group (all P<0.05).
CONCLUSIONS: Specific knockout of Med1 in T cells does not affect the development of CD4+ and CD8+ T cells in the thymus, but does affect the maintenance of peripheral CD8+ T cells. In the immune response, Med1 gene deletion affects the expression of transcription factor T-bet, which in turn to reduce Th1 cell differentiation.
目的: CD4+ T细胞在免疫应答过程中的分化受到由众多分子组成的复杂而精细的信号通路的调控，且调控T-bet表达的分子机制并未阐明。中介体复合物亚基1(mediator complex subunit 1，Med1)与多种辅助因子结合调节基因转录，促进细胞存活和增殖，影响恒定自然杀伤T细胞(invariant natural killer T cell，iNKT)的发育。本研究旨在探讨Med1对T细胞发育及CD4+ T细胞在免疫应答中分化的影响。方法: 构建T细胞特异性敲除Med1基因(KO)小鼠(Med1F/FCD4cre+)并对其进行验证。采用流式细胞术检测KO组和对照(Con)组(Med1F/FCD4cre-)小鼠胸腺、脾和淋巴结中CD4+和CD8+ T细胞的百分比及数目；用淋巴细胞脉络丛脑膜炎病毒(lymphocytic choriomeningitis virus，LCMV)感染小鼠8 d后，检测小鼠脾中CD4+ T细胞及抗原特异性(GP66+)CD4+ T细胞的百分比及数目、Th1细胞(Ly6c+PSGL1+)占CD4+ T细胞及抗原特异性CD4+ T细胞的百分比及数目、CD4+ T细胞及抗原特异性CD4+ T细胞中T-bet的荧光强度。结果: KO组与Con组小鼠胸腺中CD4+和CD8+ T细胞的百分比及数目、脾和淋巴结中CD4+ T细胞的百分比及数目差异均无统计学意义(均P>0.05)，但KO组脾和淋巴结中CD8+ T细胞的百分比及数目较Con组均显著降低(均P<0.05)。感染LCMV后，KO组与Con组小鼠脾中CD4+ T细胞及抗原特异性CD4+ T细胞的百分比及数目差异均无统计学意义(均P>0.05)，但KO组Th1细胞占CD4+ T细胞及抗原特异性CD4+ T细胞的百分比及数目、CD4+ T细胞及抗原特异性CD4+ T细胞中T-bet的表达较Con组均显著降低(均P<0.05)。结论: T细胞中特异性敲除Med1基因不影响胸腺中CD4+和CD8+ T细胞的发育，但是影响外周CD8+ T细胞的维持。在免疫应答中Med1基因缺失影响转录因子T-bet的表达，最终导致CD4+ T细胞向Th1细胞分化减少。.