in-vitro-differentiation-naive-cd4-t-cells-into-pathogenic-th17-cells

In Vitro Differentiation of Naive CD4<sup>+</sup> T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

Zhongda Hospital Affiliated to Southeast University

Southeast University

Innate lymphoid cells (ILCs) are a population of lymphoid cells that do not express T cell or B cell antigen-specific receptors. They are largely tissue-resident and enriched at mucosal sites to play a protective role against pathogens. ILCs mimic the functions of CD4 T helper (Th) subsets. Type 1 innate lymphoid cells (ILC1s) are defined by the expression of signature cytokine IFN-γ and the master transcription factor T-bet, involving in the type 1 immune response; ILC2s are characterized by the expression of signature cytokine IL-5/IL-13 and the master transcription factor GATA3, participating in the type 2 immune response; ILC3s are RORγt-expressing cells and are capable of producing IL-22 and IL-17 to maintain intestinal homeostasis. The discovery and investigation of ILCs over the past decades extends our knowledge beyond classical adaptive and innate immunology. In this review, we will focus on the roles of ILCs in intestinal inflammation and related disorders.

Innate Lymphoid Cells and Intestinal Inflammatory Disorders.

Adaptive CD4 T helper cells and their innate counterparts, innate lymphoid cells, utilize an identical set of transcription factors (TFs) for their differentiation and functions. However, similarities and differences in the induction of these TFs in related lymphocytes are still elusive. Here, we show that T helper-1 (Th1) cells and natural killer (NK) cells displayed distinct epigenomes at the Tbx21 locus, which encodes T-bet, a critical TF for regulating type 1 immune responses. The initial induction of T-bet in NK precursors was dependent on the NK-specific DNase I hypersensitive site Tbx21-CNS-3, and the expression of the interleukin-18 (IL-18) receptor; IL-18 induced T-bet expression through the transcription factor RUNX3, which bound to Tbx21-CNS-3. By contrast, signal transducer and activator of transcription (STAT)-binding motifs within Tbx21-CNS-12 were critical for IL-12-induced T-bet expression during Th1 cell differentiation both in vitro and in vivo. Thus, type 1 innate and adaptive lymphocytes utilize distinct enhancer elements for their development and differentiation.

Differential regulation of transcription factor T-bet induction during NK cell development and T helper-1 cell differentiation.

T-lymphocytes play crucial roles for maintaining immune homeostasis by fighting against various pathogenic microorganisms and establishing self-antigen tolerance. They will go through several stages and checkpoints in the thymus from progenitors to mature T cells, from CD4CD8 double negative (DN) cells to CD4CD8 double positive (DP) cells, finally become CD4 or CD8 single positive (SP) cells. The mature SP cells then emigrate out of the thymus and further differentiate into distinct subsets under different environment signals to perform specific functions. Each step is regulated by various transcriptional regulators downstream of T cell receptors (TCRs) that have been extensively studied both  and  multiple mouse models and advanced techniques, such as single cell RNA sequencing (scRNA-seq) and Chromatin Immunoprecipitation sequencing (ChIP-seq). This review will summarize the transcriptional regulators participating in the early stage of T cell development reported in the past decade, trying to figure out cascade networks in each process and provide possible research directions in the future.

Transcriptional Regulation of Early T-Lymphocyte Development in Thymus.

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors.

Transcription factors TCF-1 and GATA3 are key factors for the epigenetic priming of early innate lymphoid progenitors toward distinct cell fates.

CD8 T cells belonging to the adaptive immune system play key roles in defending against viral infections and cancers. The current CD8 T cell-based immunotherapy has emerged as a superior therapeutic avenue for the eradication of tumor cells and long-term prevention of their recurrence in hematologic malignancies. It is believed that an effective adaptive immune response critically relies on the help of the innate compartment. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that have been considered some of the first cells to respond to infections and can secrete a large amount of diverse cytokines and chemokines to widely modulate the innate and adaptive immune responders. Like CD8 T cells, iNKT cells also play an important role in defense against intracellular pathogenic infections and cancers. In this review, we will discuss the CD8 T-cell immunity contributed by iNKT cells, including iNKT cell-mediated cross-priming and memory formation, and discuss recent advances in our understanding of the mechanisms underlying memory CD8 T-cell differentiation, as well as aging-induced impairment of T-cell immunity.

CD8 T-cell immunity orchestrated by iNKT cells.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues.

Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a key molecule in T-cell development and has been linked to immune diseases. However, its role in antitumour CD8T cell immunity remains unclear. Here, we demonstrated that Tespa1 plays an important role in antitumour CD8T cell immunity. First, compared with wild-type (WT) mice, Lewis lung cancer cells grew faster in Tespa1 knockout (Tespa1) mice, with reduced apoptosis, and decreased CD8T cells in peripheral blood and tumor tissues. Second, the proportion of CD8T and Th1 cells in the splenocytes of Tespa1 mice was lower than that in WT mice. Third, Tespa1 CD8 tumor-infiltrating lymphocytes (TILs) showed weakened proliferation, invasion, cytotoxicity, and protein expression of IL-2 signalling pathway components compared to WT CD8TILs. Furthermore, PD-1 expression in CD8TILs was higher in Tespa1 than in WT mice. Lastly, CD8TILs in WT mice improved the antitumour ability of Tespa1 mice. In conclusion, these findings suggest that Tespa1 plays a critical role in the tumor immune system by regulating CD8T cells.

Tespa1 deficiency reduces the antitumour immune response by decreasing CD8T cell activity in a mouse Lewis lung cancer model.

Over the past few decades, food allergy has continued to rise, significantly affecting our health, economy, and quality of life. However, current therapeutic strategies have limited efficacy and need to be improved. One alternative to prevent or reduce allergies is to modulate immunity and microbiota. Human milk (HM) could be considered a protective factor against food allergy, but how probiotics in human milk impact the susceptibility to food allergy remains unknown. Therefore, we studied the preventive impact of human milk  Probio-M9 on food allergy in ovalbumin (OVA)-sensitized mice. We studied the effects of oral administration of Probio-M9 on allergic signatures, immune response, gut microbiota, and metabolism. Oral therapeutic administration of live Probio-M9, but not heat-killed Probio-M9, significantly reduces OVA-specific IgE (OVA-sIgE), histamine, and mMCP-1 (mouse mast cell protease-1) levels in OVA-sensitized mice. Moreover, Probio-M9 supplementation reduced allergic inflammation and changes in the Th2/Th1 balance toward a dampened Th2 response. 16S rDNA sequencing analysis revealed an increased ratio of / (F/B) and the relative abundance of short-chain fatty acid (SCFA)-producing  in the feces after Probio-M9 intake. Simultaneously, Probio-M9 significantly increased the levels of SCFAs and promoted the phosphorylation of signal transducer and activator of transcription 3 (STAT3), thereby inducing the expression of the antimicrobial peptides (AMPs) Reg3b and Reg3g. Our findings suggest that the use of Probio-M9 can be a potent strategy in food allergy prevention.

 Probio-M9 alleviates OVA-sensitized food allergy through modulating gut microbiota and its metabolism.

Innate lymphoid cells (ILCs) in the lamina propria of the small intestine (siLP) are essential for maintaining intestinal immune homeostasis; however, their isolation remains challenging. Here, we present a protocol for the rapid isolation of siLP ILCs. We describe steps for small intestine collection and trimming, epithelial cell dissociation, lamina propria digestion, and ILC analysis by flow cytometry. For complete details on the use and execution of this protocol, please refer to Zheng et al..

Article	Year
Innate Lymphoid Cells and Intestinal Inflammatory Disorders. International journal of molecular sciences\| PubMed ID: 35163778	2022
Differential regulation of transcription factor T-bet induction during NK cell development and T helper-1 cell differentiation. Immunity\| PubMed ID: 35381213	2022
Transcriptional Regulation of Early T-Lymphocyte Development in Thymus. Frontiers in immunology\| PubMed ID: 35432347	2022
Transcription factors TCF-1 and GATA3 are key factors for the epigenetic priming of early innate lymphoid progenitors toward distinct cell fates. Immunity\| PubMed ID: 35882235	2022
CD8 T-cell immunity orchestrated by iNKT cells. Frontiers in immunology\| PubMed ID: 36741397	2022
Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues. Cell reports\| PubMed ID: 37540600	2023
Tespa1 deficiency reduces the antitumour immune response by decreasing CD8T cell activity in a mouse Lewis lung cancer model. International immunopharmacology\| PubMed ID: 37660596	2023
Probio-M9 alleviates OVA-sensitized food allergy through modulating gut microbiota and its metabolism. Food & function\| PubMed ID: 37982421	2023
Protocol for isolating innate lymphoid cells from murine lamina propria of the small intestine. STAR protocols\| PubMed ID: 39383042	2024

Mingzhu Zheng

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