Long noncoding RNA LIRIL2R modulates FOXP3 levels and suppressive function of human CD4+ regulatory T cells by regulating IL2RA.

Regulatory T cells (Tregs) are central in controlling immune responses, and dysregulation of their function can lead to autoimmune disorders or cancer. Despite extensive studies on Tregs, the basis of epigenetic regulation of human Treg development and function is incompletely understood. Long intergenic noncoding RNAs (lincRNA)s are important for shaping and maintaining the epigenetic landscape in different cell types. In this study, we identified a gene on the chromosome 6p25.3 locus, encoding a lincRNA, that was up-regulated during early differentiation of human Tregs. The lincRNA regulated the expression of interleukin-2 receptor alpha (IL2RA), and we named it the lincRNA regulator of IL2RA (LIRIL2R). Through transcriptomics, epigenomics, and proteomics analysis of LIRIL2R-deficient Tregs, coupled with global profiling of LIRIL2R binding sites using chromatin isolation by RNA purification, followed by sequencing, we identified IL2RA as a target of LIRIL2R. This nuclear lincRNA binds upstream of the IL2RA locus and regulates its epigenetic landscape and transcription. CRISPR-mediated deletion of the LIRIL2R-bound region at the IL2RA locus resulted in reduced IL2RA expression. Notably, LIRIL2R deficiency led to reduced expression of Treg-signature genes (e.g., FOXP3, CTLA4, and PDCD1), upregulation of genes associated with effector T cells (e.g., SATB1 and GATA3), and loss of Treg-mediated suppression.

HIC1 interacts with FOXP3 multi protein complex: Novel pleiotropic mechanisms to regulate human regulatory T cell differentiation and function.

Transcriptional repressor, hypermethylated in cancer 1 (HIC1) participates in a range of important biological processes, such as tumor repression, immune suppression, embryonic development and epigenetic gene regulation. Further to these, we previously demonstrated that HIC1 provides a significant contribution to the function and development of regulatory T (Treg) cells. However, the mechanism by which it regulates these processes was not apparent. To address this question, we used affinity-purification mass spectrometry to characterize the HIC1 interactome in human Treg cells. Altogether 61 high-confidence interactors were identified, including IKZF3, which is a key transcription factor in the development of Treg cells. The biological processes associated with these interacting proteins include protein transport, mRNA processing, non-coding (ncRNA) transcription and RNA metabolism. The results revealed that HIC1 is part of a FOXP3-RUNX1-CBFB protein complex that regulates Treg signature genes thus improving our understanding of HIC1 function during early Treg cell differentiation.

The Transcription Factor MAZR/PATZ1 Regulates the Development of FOXP3+ Regulatory T Cells.

Forkhead box protein P3+ (FOXP3+) regulatory T cells (Treg cells) play a key role in maintaining tolerance and immune homeostasis. Here, we report that a T cell-specific deletion of the transcription factor MAZR (also known as PATZ1) leads to an increased frequency of Treg cells, while enforced MAZR expression impairs Treg cell differentiation. Further, MAZR expression levels are progressively downregulated during thymic Treg cell development and during in-vitro-induced human Treg cell differentiation, suggesting that MAZR protein levels are critical for controlling Treg cell development. However, MAZR-deficient Treg cells show only minor transcriptional changes ex vivo, indicating that MAZR is not essential for establishing the transcriptional program of peripheral Treg cells. Finally, the loss of MAZR reduces the clinical score in dextran-sodium sulfate (DSS)-induced colitis, suggesting that MAZR activity in T cells controls the extent of intestinal inflammation. Together, these data indicate that MAZR is part of a Treg cell-intrinsic transcriptional network that modulates Treg cell development.

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells.

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function.

Ca2+-dependent Focal Exocytosis of Golgi-derived Vesicles Helps Phagocytic Uptake in Macrophages.

The role of Golgi apparatus during phagocytic uptake by macrophages has been ruled out in the past. Notably, all such reports were limited to Fcγ receptor-mediated phagocytosis. Here, we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos, except the IgG-coated ones. We report recruitment of mannosidase-II-positive Golgi-derived vesicles during uptake of diverse targets, including latex beads, Escherichia coli, Salmonella typhimurium, and Mycobacterium tuberculosis in human and mouse macrophages. The recruitment of mannosidase-II vesicles was an early event mediated by focal exocytosis and coincided with the recruitment of transferrin receptor, VAMP3, and dynamin-2. Brefeldin A treatment inhibited mannosidase-II recruitment and phagocytic uptake of serum-coated or -uncoated latex beads and E. coli However, consistent with previous studies, brefeldin A treatment did not affect uptake of IgG-coated latex beads. Mechanistically, recruitment of mannosidase-II vesicles during phagocytic uptake required Ca2+ from both extra- and intracellular sources apart from PI3K, microtubules, and dynamin-2. Extracellular Ca2+ via voltage-gated Ca2+ channels established a Ca2+-dependent local phosphatidylinositol 1,4,5-trisphosphate gradient, which guides the focal movement of Golgi-derived vesicles to the site of uptake. We confirmed Golgi-derived vesicles recruited during phagocytosis were secretory vesicles as their recruitment was sensitive to depletion of VAMP2 or NCS1, whereas recruitment of the recycling endosome marker VAMP3 was unaffected. Depletion of both VAMP2 and NCS1 individually resulted in the reduced uptake by macrophages. Together, the study provides a previously unprecedented role of Golgi-derived secretory vesicles in phagocytic uptake, the key innate defense function.