Transcription Factor RUNX3 Mediates Plasticity of ThGM Cells Toward Th1 Phenotype

Abstract

GM-CSF-producing T helper (Th) cells play a crucial role in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Recent studies have identified a distinct population of GM-CSF-producing Th cells, named ThGM cells, that also express cytokines TNF, IL-2, and IL-3, but lack expression of master transcription factors (TF) and signature cytokines of commonly recognized Th cell lineages. ThGM cells are highly encephalitogenic in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Similar to Th17 cells, in response to IL-12, ThGM cells upregulate expression of T-bet and IFN-γ and switch their phenotype to Th1. Here we show that in addition to T-bet, TF RUNX3 also contributes to the Th1 switch of ThGM cells. T-bet-deficient ThGM cells in the CNS of mice with EAE had low expression of RUNX3, and knockdown of RUNX3 expression in ThGM cells abrogated the Th1-inducing effect of IL-12. Comparison of ThGM and Th1 cell transcriptomes showed that ThGM cells expressed a set of TFs known to inhibit the development of other Th lineages. Lack of expression of lineage-specific cytokines and TFs by ThGM cells, together with expression of TFs that inhibit the development of other Th lineages, suggests that ThGM cells are a non-polarized subset of Th cells with lineage characteristics.

Oxidized low‐density lipoproteins impair the pro‐atherosclerotic effect of granulocyte‐macrophage‐colony‐stimulating factor‐producing T helper cells on macrophages

“…To corroborate the identity of ThGM cells, we quantified the mRNA levels of Twist‐related protein 1 (TWIST1), transcriptional regulating factor 1 (TRERF1), musculin (MSC) and peroxisome proliferator‐activated receptor gamma (PPARγ), all of which are ThGM‐associated transcription factors 22 . As indicated in Figure S4, ThGM cells (i.e.…”

“…Meanwhile, we found that live aortic ThGM cells were enriched in CD4 + CCR6 − CCR8 − CXCR3 − CCR10 + T cells, consistent with former reports. 10,22 We are the first to report the existence of ThGM cells in atherosclerotic aortas. Notably, ThGM-associated transcription factors such as TWIST1, TRERF1, MSC and PPARγ were highly expressed by these cells.…”

“…However, they might also originate from ThGM cells since ThGM cells could turn into Th1 cells. 22 Although we regarded subset 'b' and subset 'a' as Th1 cells to facilitate analysis, their true identities deserve future investigations.…”

“…Naïve CD4 + CD25 − T cells were collected from normal C57BL/6J mouse spleens using the MagniSort™ mouse CD4 Naïve T cell enrichment kit (Invitrogen) following the vendor's manual. ThGM differentiation was conducted based on previous reports 12,22,23 . Briefly, 2.5 × 10 5 CD4 + CD25 − T cells were resuspended in supplemented RPMI 1640 and placed in each well of a 96‐well microplate (Corning), followed by stimulation with 5 μg/mL plate‐bound CD3ε antibody (145‐2C11, eBioscience), 2 μg/mL soluble CD28 antibody (37.51, eBioscience), 2 ng/mL mouse IL‐7 (407‐ML‐025/CF, R&D Systems), 10 μg/mL IL‐12 antibody (AF‐419‐NA, R&D Systems), 10 μg/mL IFN‐γ antibody (MAB485‐SP, R&D Systems) and 5 μg/mL IL‐4 antibody (AF‐404‐SP, R&D Systems) for consecutive 3 days.…”

“…ThGM differentiation was conducted based on previous reports. 12,22,23 Briefly, 2.5 × 10 5 CD4 + CD25 − T cells were resuspended in supplemented RPMI 1640 and placed in each well of a 96-well microplate (Corning), followed by stimulation with 5 μg/mL plate-bound CD3ε antibody (145-2C11, eBioscience), 2 μg/ mL soluble CD28 antibody (37.51, eBioscience), 2 ng/mL mouse IL-7 (407-ML-025/CF, R&D Systems), 10 μg/mL IL-12 antibody (AF-419-NA, R&D Systems), 10 μg/mL IFN-γ antibody (MAB485-SP, R&D Systems) and 5 μg/mL IL-4 antibody (AF-404-SP, R&D Systems) for consecutive 3 days. To evaluate the effects of oxLDL and native LDL on ThGM differentiation, oxLDL (Cat# L34357, Invirtogen) or native LDL (Cat# L3486, Invirtogen) was added at the start of the culture at a concentration of 50 μg/mL.…”