Genome-wide screening identifies TRIM33 as an essential regulator of dendritic cell differentiation

Background

Dendritic cells (DCs) serve as a bridge between innate and adaptive immunity by recognizing pathogens through pattern recognition receptors (such as TLRs) and modulating antigen-specific T cell responses. DCs are mainly divided into two types: plasmacytoid dendritic cells (pDCs), which produce interferons, and conventional dendritic cells (cDCs), which present antigens. pDCs recognize pathogen-derived nucleic acids through endosomal TLRs (TLR7 and TLR9) and rapidly produce type I interferons and other cytokines, whereas cDCs have high levels of major histocompatibility complex (MHC) class II molecules and antigen-presenting machinery that can efficiently activate naive antigen-specific T cells. cDCs are further divided into CDC1, which can cross-present antigens to CD8+ T cells, and CDC2, which specialize in presenting exogenous antigens to CD4+ T cells.

All DCs are short-lived hematopoietic cells continuously generated in the bone marrow driven by Flt3 ligand (FLT3L). The receptor FLT3 is expressed on multipotent hematopoietic progenitor cells and all mature DCs. However, the precise molecular mechanisms driven by Flt3L signals are still not fully understood, and how these signals balance progenitor cell proliferation and DC differentiation remains an unsolved mystery.

Source of the Paper

This paper was written by Ioanna Tiniakou and colleagues, with team members from institutions such as New York University Grossman School of Medicine and Humboldt Universität zu Berlin. It was published on April 12, 2024, in the journal “Science Immunology” with the title “Genome-wide screening identifies TRIM33 as an essential regulator of dendritic cell differentiation.”

Research Process

Genome-wide Screening

To explore the mechanisms of DC differentiation driven by FLT3L, researchers used conditionally immortalized Hoxb8-FL cell lines. These cells remain undifferentiated in the presence of estrogen and FLT3L and can produce all myeloid cells and DC subsets. By removing estrogen and culturing in the presence of FLT3L, Hoxb8-FL cells can differentiate into functional pDCs and cDCs within 7 days.

Researchers performed CRISPR-Cas9 gene knockout screening using the “BriE” sgRNA library, targeting approximately 19,600 mouse genes. The transduced cells were expanded under FLT3L and estrogen conditions to obtain initial progenitor cell samples, then removed estrogen to differentiate into DC subsets. Finally, the researchers analyzed the sgRNA content of each sample through genomic sequencing, using the “crisptimer” algorithm to identify gene knockout mutants.

The screening results indicated that multiple genes play significant roles in DC differentiation, including subunits of the TSC and GATOR1 complexes, which promote DC differentiation by inhibiting mTOR signaling to limit progenitor cell proliferation. The study also identified TRIM33, a transcriptional repressor, as a crucial regulator of DC differentiation. Conditional in vivo targeting experiments in mice showed that deleting TRIM33 led to a significant reduction in all DC subsets, including pDCs and cross-presenting CDC1 subsets, without affecting monocytes or granulocytes.

Further Validation

Researchers further explored the role of mTOR signaling in DC differentiation. The results showed that TSC and GATOR1 complexes act as “switches” by limiting mTOR activity to regulate the transition between progenitor cell expansion and DC differentiation driven by FLT3L. By using mTOR inhibitors Torin1 and rapamycin, researchers validated that these inhibitors completely suppressed the growth of undifferentiated Hoxb8-FL cells at different concentrations without inhibiting DC differentiation.

Finally, the research team used another sgRNA library for transcription factor screening to further confirm regulators of FLT3L-driven DC differentiation, especially TRIM33. Through in vivo experiments with conditionally Trim33-deleted mice, researchers confirmed that Trim33 is an essential regulator for all major DC subsets.

Research Results

1. Identification of Gene Regulators: Genome-wide screening identified several genes regulating FLT3L-driven DC differentiation, including subunits of the TSC and GATOR1 complexes, which promote DC differentiation by inhibiting mTOR signaling. Additionally, TRIM33 was found to play a critical role in DC differentiation.

2. In Vivo Validation: In mouse models, conditional deletion of Trim33 was observed to significantly reduce all DC subsets without affecting monocytes or granulocytes.

3. mTOR Signaling Study: Further investigation into mTOR signaling revealed that TSC and GATOR1 complexes regulate progenitor cell expansion and DC differentiation by limiting mTOR activity.

4. Transcription Factor Screening: Additional sgRNA library screening further confirmed the key role of TRIM33 in FLT3L-driven DC differentiation.

Research Conclusions

The study elucidated the molecular mechanisms of FLT3L-driven DC differentiation, particularly the role of TSC and GATOR1 in regulating mTOR signaling as a “switch.” The study also confirmed TRIM33 as a critical regulator of DC differentiation, highlighting its importance in the process. These findings provide important insights into how FLT3L signals differentiate dendritic cells and other myeloid cells.

Significance and Value of the Study

1. Scientific Significance: The study identified several key genes and their roles in FLT3L-driven DC differentiation, revealing the molecular mechanisms of DC differentiation, especially the critical role of mTOR signaling.

2. Applied Value: TRIM33 and other identified regulatory genes provide potential targets for future therapies aimed at modulating DC differentiation, having significant clinical application value.

3. Innovativeness: The study used genome-wide CRISPR-Cas9 screening to identify key regulatory factors from a broad gene spectrum while validating these findings through conditional gene knockout mouse models.

This study provides new insights into dendritic cell differentiation and points out important directions for future research and clinical applications.