Natural killer (NK) cells are innate lymphocytes responsible for early defense against infections and malignant cells. Specific transcription factors crucial for NK cell development and function include PRDM1, which also regulates T cell homeostasis and is essential for the terminal differentiation of B cells into plasma cells. Importantly, PRDM1 deletion, methylation and loss-of-function mutations were commonly detected in NK cell malignancies. However, the detailed mechanisms through which PRDM1 regulates NK cell homeostasis are still largely undefined.
Here, we employed an in vitro culture system of human NK cells isolated from healthy donors, in which the NK cells were cultured in the presence of IL-2 with or without an engineered feeder cell line, K562-Cl9-mb21, expressing membrane-bound IL-21, 4-1BBL and CD86. The NK cells were able to expand for months when co-cultured with feeder cells, whereas IL-2 alone could only maintain NK cell survival with limited proliferation for one week.
We performed ChIP-seq to compare the genome-wide binding profiles of PRDM1 in NK cells grown with or without feeder cells. We found that PRDM1 bound much fewer target genes (802) in NK cells with feeder than in NK cells with IL-2 alone (2880), and 98.5% (790/802) of the binding sites in feeder-stimulated NK cells overlapped with those found in NK cells without feeder. The PRDM1 consensus motifs were almost identical in both conditions. MEME analyses also identified motifs of other transcription factors enriched in the PRDM1 binding sites, such as the RUNX and T-Box families. Interestingly, the RUNX and T-Box motifs, among others, were more enriched in the PRDM1 binding sites lacking the PRDM1 motif than in those with the PRDM1 motif, which indicates that PRDM1 may often bind to DNA indirectly through other transcription factors.
PRDM1 bound a large number of genes in the NK or T cell receptor signaling pathway, which are employed during NK cell activation. In addition, several genes encoding immune checkpoints that may restrict NK cell activation such as TIGIT, HAVCR2 (TIM3), and IL-1R8 were targeted by PRDM1. Many genes encoding NK cell inhibitory and activating receptors were also bound by PRDM1. Moreover, PRDM1 was found to target transcription factors that are important for NK cell development and homeostasis, such as the RUNX family, TBX21, MAF, and PRDM1 itself. Despite an extensive overlap of PRDM1 binding sites detected in NK cells grown with or without feeder cells, the most enriched pathways were not exactly the same. Importantly, our RNA-seq data on PRDM1-knockout NK cells validated the regulatory role of PRDM1 on a fraction of these PRDM1-bound genes and interestingly, PRDM1 appeared to be an activator for some of the genes, including those encoding immune checkpoint molecules TIGIT, HAVCR2, and IL-1R8.
We also utilized ATAC-seq to examine the chromatin accessibility of NK cells grown with feeder cells or with IL-2 alone. We identified differentially enriched pathways for the NK cells cultured under different conditions. When compared with the PRDM1 ChIP-seq, we found that those PRDM1 binding sites that contain a consensus PRDM1 motif were less likely to be accessible to transposase than those without the PRDM1 motif, thereby confirming the transcriptional repressor role of PRDM1.
To further understand how PRDM1 regulates its target genes in NK cells, we performed mass spectrometric analysis on the protein complexes associated with PRDM1. We were able to identify the interaction of PRDM1 with the corepressor Groucho (TLE3) and components from the SIN3, NCoR, and NuRD complex, which have been previously reported in mouse plasma cells. Surprisingly, the protein compositions of the PRDM1-associated complexes from the two NK cell populations were very different. Although PRDM1 is generally associated with transcriptional repression, we also detected the association of PRDM1 with transcriptional activators or coactivators, such as the RUNX-binding protein CBFβ and the T-Box family member EOMES, which may thereby upregulate some of the PRDM1 target genes.
In summary, we found that PRDM1 binds and regulates an extensive network of genes responsible for NK cell activation and function. Extrinsic stimuli, as provided by the feeder cells, can alter the extent and profile of PRDM1 binding as well as the associated protein complexes and hence alter its regulatory function in NK cells.
Disclosures
No relevant conflicts of interest to declare.
SMAD4 promotes TGF-β–independent NK cell homeostasis and maturation and antitumor immunity
