The mammalian small intestine epithelium harbors a peculiar population of CD4+CD8αα+ T cells that are derived from mature CD4+ T cells through reprogramming of lineage-specific transcription factors. CD4+CD8αα+ T cells occupy a unique niche in T cell biology because they exhibit mixed phenotypes and functional characteristics of both CD4+ helper and CD8+ cytotoxic T cells. The molecular pathways driving their generation are not fully mapped. However, recent studies demonstrate the unique role of the commensal gut microbiota as well as distinct cytokine and chemokine requirements in the differentiation and survival of these cells. We review the established and newly identified factors involved in the generation of CD4+CD8αα+ intraepithelial lymphocytes (IELs) and place them in the context of the molecular machinery that drives their phenotypic and functional differentiation.

Ghrelin regulates diverse physiological activities. However, the effects of this hormone on the milk fat synthesis remain unknown. This study aimed to investigate the effect of acylated ghrelin (AG) on milk fat synthesis by modifying the expression (knockdown or overexpression) of growth hormone secretagogue receptor 1a (GHSR1a) and Th-inducing POK (ThPOK) in primary bovine mammary epithelial cells (BMECs). The results showed that AG significantly increased the triglyceride relative content from 260.83 ± 9.87 to 541.67 ± 8.38 in BMECs via GHSR1a. ThPOK functions as a key regulatory target downstream of AG, activating the PI3K and mTOR signaling pathways to promote milk fat synthesis in BMECs. Moreover, AG-regulated ThPOK by increasing the EP300 activity, which promoted ThPOK acetylation to protect it from proteasomal degradation. In conclusion, AG increases ThPOK acetylation and stabilizes ThPOK through GHSR1a, thereby activating the PI3K/mTOR signaling pathway and ultimately promoting the milk fat synthesis in BMECs.

Insulin-like growth factor-1 (IGF-1), in addition to its classic effects on cell proliferation and organism growth, has pleiotropic actions on the immune system, particularly on the thymus. Thus, the objective of this study was to evaluate the influence of IGF-1 on molecules involved in the survival of thymocytes in vitro using a co-culture system with thymic stromal cells obtained from C57BL/6 mice. The obtained thymic stroma has contained thymic epithelial cells, macrophages, dendritic cells, fibroblasts, and preserved the expression of the major histocompatibility complex (MHC) molecules. Fresh thymocytes were added to these cultures and the co-culture were treated daily with IGF-1 (100 ng/mL) for 3 days. In this scheme, the viability of the thymocytes was about 70%, either in the control (non-treated cells) or in the IGF-1-treated cultures. It was found that IGF-1 was able to increase the percentage of thymocytes from the CD4+ single-positive (SP) subset. This result was accompanied by an increase in the MHC II expression on thymic stromal cells and an augment on the interleukin-7 receptor (CD127) on the surface of the CD4 SP thymocytes after treatment with IGF-1. Finally, IGF-1 treatment increased the expression of the ThPOK encoding gene Zbtb7b, which is involved in the differentiation of CD4+ SP thymocytes. Our study demonstrates the participation of IGF-1 in the thymocyte/thymic stroma interactions, especially in the extended survival of the CD4+ lineage in the thymus.

Innate lymphoid cells (ILCs) have been identified as a heterogeneous population of lymphocytes that mirrors the cytokine and transcriptional profile of adaptive T cells. The dynamic balance between key transcription factors determines the heterogeneity, plasticity, and functions of ILC subsets. The transcription factor ThPOK is highly conserved in biological evolution and exerts pivotal functions in the differentiation of T cells. However, the function of ThPOK in ILC3s has not been identified. Here, we found that ThPOK regulated the homeostasis of ILC3s, as mice lacking ThPOK showed decreased NKp46+ ILC3s and increased CCR6- NKp46- ILC3s. ThPOK-deficient mice were more sensitive to S. typhimurium infection due to the impaired IFN-γ secretion of NKp46+ ILC3s. Furthermore, ThPOK participates in ILC3-mediated control of C. rodentium infection by negatively regulating IL-17A secretion. ThPOK preserves the identity of NKp46+ ILC3s by repressing RORγt, which indirectly releases T-bet expression. On the molecular level, ThPOK directly binds to Rorc and Il23r to restrain their expression which further modulates IL-17A secretion. Collectively, our analysis revealed a critical role of ThPOK in the homeostasis and functions of ILC3 subsets.

Gastric cancer is the second most frequently diagnosed cancer worldwide. Weak immunogenicity helps cancer cells escape from immune elimination and grow into predominant subpopulations. This study aimed to investigate the effect of Zinc finger and BTB domain containing 7B (Zbtb7b, Alias ThPOK) on T cell activation after coculture with gastric cancer cells.
Cell Counting Kit-8 assay (CCK-8) was performed to explore the viability of gastric cancer cells. Flow cytometry analysis was used to measure CD3+ T cell proliferation and the ratio of activated IFN-γ+ T cells which were co-incubated with gastric cancer cells (HGC-27, SNU-1). The binding between ThPOK and the promoter of its target sperm tail PG-rich repeat containing 1 (STPG1) was explored using ChIP and luciferase reporter assays. Relative gene expression was quantified using RT-qPCR.
ThPOK was expressed at a low level in gastric cancer tissues and cells at mRNA and protein levels. Gastric cancer patients with lower ThPOK expression had poorer prognosis. ThPOK overexpression suppressed gastric cancer cell viability and increased T cell activation. ThPOK served as a transcription factor for STPG1. STPG1 expression was also at a low level in the tissues and cells of gastric cancer. ThPOK positively regulated the mRNA and protein levels of STPG1 in gastric cancer cells. Moreover, ThPOK was demonstrated to bind with STPG1 promoter. STPG1 upregulation also exerted inhibitory effects on gastric cancer cell viability and T cell activation. Additionally, ThPOK and STPG1 were revealed to inactivate the ERK pathway in gastric cancer cells.
ThPOK inhibits gastric cancer cell viability and increases T cell activation by inducing STPG1 to inactivate the ERK pathway.

Gastric cancer (GC), originated from gastric mucosa, is a malignant tumor causing numerous deaths globally. The present study used the coculture of T cells with supernatant of the GC cells (HGC-27, SNU-1) and investigated the function and regulatory mechanism of Zinc finger and BTB domain containing 7B (ZBTB7B, alias ThPOK) on T cell proliferation. Flow cytometry analysis was used to measure the proliferation of CD3+ T cells and IFN-γ+ T cells. We found that low level of ThPOK was associated with poor prognosis in GC patients. ThPOK was lowly expressed in GC cells at the mRNA and protein levels. ThPOK overexpression inhibited GC cell viability and promoted proliferation of T cells. ThPOK was identified to function as a transcription factor for TNFRSF12A. TNFRSF12A was upregulated in GC tissues and cells and high level of TNFRSF12A was associated with poor prognosis in GC patients. ThPOK knockdown elevated TNFRSF12A level in GC cells. ThPOK was revealed to bind with the promoter of TNFRSF12A. TNFRSF12A silencing also inhibited GC cell viability and promoted T cell activation and proliferation. Additionally, ThPOK was demonstrated to inactivate the NF-kB pathway by downregulating TNFRSF12A in GC cells. Overall, ThPOK suppresses cell viability in GC and increases the activation and proliferation of T cells by targeting TNFRSF12A to inactivate the NF-kB pathway.

αβ lineage T cells, most of which are CD4+ or CD8+ and recognize MHC I- or MHC II-presented antigens, are essential for immune responses and develop from CD4+CD8+ thymocytes. The absence of in vitro models and the heterogeneity of αβ thymocytes have hampered analyses of their intrathymic differentiation. Here, combining single-cell RNA and ATAC (chromatin accessibility) sequencing, we identified mouse and human αβ thymocyte developmental trajectories. We demonstrated asymmetric emergence of CD4+ and CD8+ lineages, matched differentiation programs of agonist-signaled cells to their MHC specificity, and identified correspondences between mouse and human transcriptomic and epigenomic patterns. Through computational analysis of single-cell data and binding sites for the CD4+-lineage transcription factor Thpok, we inferred transcriptional networks associated with CD4+- or CD8+-lineage differentiation, and with expression of Thpok or of the CD8+-lineage factor Runx3. Our findings provide insight into the mechanisms of CD4+ and CD8+ T cell differentiation and a foundation for mechanistic investigations of αβ T cell development.

CD4+ and CD8+ T cells are dichotomous lineages in adaptive immunity. While conventionally viewed as distinct fates that are fixed after thymic development, accumulating evidence indicates that these two populations can exhibit significant lineage plasticity, particularly upon TCR-mediated activation. We define a novel CD4-CD8αβ+ MHC II-recognizing population generated by lineage conversion from effector CD4+ T cells. CD4-CD8αβ+ effector T cells downregulated the expression of T helper cell-associated costimulatory molecules and increased the expression of cytotoxic T lymphocyte-associated cytotoxic molecules. This shift in functional potential corresponded with a CD8+-lineage skewed transcriptional profile. TCRβ repertoire sequencing and in vivo genetic lineage tracing in acutely infected wild-type mice demonstrated that CD4-CD8αβ+ effector T cells arise from fundamental lineage reprogramming of bona fide effector CD4+ T cells. Impairing autophagy via functional deletion of the initiating kinase Vps34 or the downstream enzyme Atg7 enhanced the generation of this cell population. These findings suggest that effector CD4+ T cells can exhibit a previously unreported degree of skewing towards the CD8+ T cell lineage, which may point towards a novel direction for HIV vaccine design.

The generation of high-affinity neutralizing antibodies, the objective of most vaccine strategies, occurs in B cells within germinal centers (GCs) and requires rate-limiting \"help\" from follicular helper CD4+ T (Tfh) cells. Although Tfh differentiation is an attribute of MHC II-restricted CD4+ T cells, the transcription factors driving Tfh differentiation, notably Bcl6, are not restricted to CD4+ T cells. Here, we identified a requirement for the CD4+-specific transcription factor Thpok during Tfh cell differentiation, GC formation, and antibody maturation. Thpok promoted Bcl6 expression and bound to a Thpok-responsive region in the first intron of Bcl6. Thpok also promoted the expression of Bcl6-independent genes, including the transcription factor Maf, which cooperated with Bcl6 to mediate the effect of Thpok on Tfh cell differentiation. Our findings identify a transcriptional program that links the CD4+ lineage with Tfh differentiation, a limiting factor for efficient B cell responses, and suggest avenues to optimize vaccine generation.

Memory CD4+ T cells mediate long-term immunity, and their generation is a key objective of vaccination strategies. However, the transcriptional circuitry controlling the emergence of memory cells from early CD4+ antigen-responders remains poorly understood. Here, using single-cell RNA-seq to study the transcriptome of virus-specific CD4+ T cells, we identified a gene signature that distinguishes potential memory precursors from effector cells. We found that both that signature and the emergence of memory CD4+ T cells required the transcription factor Thpok. We further demonstrated that Thpok cell-intrinsically protected memory cells from a dysfunctional, effector-like transcriptional program, similar to but distinct from the exhaustion pattern of cells responding to chronic infection. Mechanistically, Thpok- bound genes encoding the transcription factors Blimp1 and Runx3 and acted by antagonizing their expression. Thus, a Thpok-dependent circuitry promotes both memory CD4+ T cells\' differentiation and functional fitness, two previously unconnected critical attributes of adaptive immunity.