The Long Non-coding RNA Flatr Anticipates Foxp3 Expression in Regulatory T Cells

Abstract

Mammalian genomes encode a plethora of long non-coding RNA (lncRNA). These transcripts are thought to regulate gene expression, influencing biological processes from development to pathology. Results from the few lncRNA that have been studied in the context of the immune system have highlighted potentially critical functions as network regulators. Here we explored the nature of the lncRNA transcriptome in regulatory T cells (Tregs), a subset of CD4⁺ T cells required to establish and maintain immunological self-tolerance. The identified Treg lncRNA transcriptome showed distinct differences from that of non-regulatory CD4⁺ T cells, with evidence of direct shaping of the lncRNA transcriptome by Foxp3, the master transcription factor driving the distinct mRNA profile of Tregs. Treg lncRNA changes were disproportionally reversed in the absence of Foxp3, with an enrichment for colocalisation with Foxp3 DNA binding sites, indicating a direct coordination of transcription by Foxp3 independent of the mRNA coordination function. We further identified a novel lncRNA Flatr, as a member of the core Treg lncRNA transcriptome. Flatr expression anticipates Foxp3 expression during in vitro Treg conversion, and Flatr-deficient mice show a mild delay in in vitro and peripheral Treg induction. These results implicate Flatr as part of the upstream cascade leading to Treg conversion, and may provide clues as to the nature of this process.

Keywords: Flatr; Foxp3; Tregs; iTreg induction; lncRNA.

Figure 1

Treg lncRNA transcriptome is shaped by Foxp3 expression. (A) Volcano-plot showing differential expression of 1765 lncRNA in naïve CD4⁺ T cells (CD4⁺CD62L⁺CD44⁻GFP⁻) compared to Tregs (CD4⁺GFP⁺) from Foxp3GFP mice (n = 3 replicates from pooled biological samples). Flatr annotated and marked in green. Previously published Flicr annotated. Downregulated (blue). Upregulated (red). P < 0.05 cutoff for differential expression. (B) log2 fold change expression between naïve CD4⁺ T cells and Tregs, comparing thymic and peripheral subsets [(57), note only polyadenylated lncRNA present in database]. (C) Expression of selected, Treg-specific lncRNAs within thymic subsets of T cell development and peripheral naïve CD4⁺ T cells and Tregs [(57), note only polyadenylated lncRNA present in database]. (D) Non differential, core Treg upregulated, and core Treg downregulated lncRNAs grouped by genomic location relative to protein-coding genes [sense, antisense, and long intergenic non coding (lincRNA)]. (E) Foxp3 Chip-seq peaks (GSE40686) within the promoter region or the gene body of non-differential expressed lncRNAs or core Treg lncRNAs. (F) Differential expression of core lncRNAs in Foxp3⁺ Treg and Foxp3KIKO Treg (GSE40686).

Figure 2

Flatr is a Tre-specific intronic lncRNA that anticipates Foxp3 expression. (A) Genomic plot showing location of murine Cwc27 with intronic location of Flatr and RNA-seq read data, visualized with IGV. (B) Sequence conservation across mouse, human, and chimpanzee. Regulatory region, annotated by Ensemble (ENSMUSR00000080103), in exon 2 is highlighted (yellow). Exons are marked by black bars. Regions of more than 100bp and with more than 70% similarity are marked (orange). (C) Flatr and (D) Cwc27 expression as FPKM values in naïve and Treg cells as determined by RNAseq analysis. Each point is an individual mouse. N = 3/group. FPKM, fragments per kilobase per million. (E) Flatr expression as determined in the T cell Th-express compendium (29). (F) RT-qPCR analysis on FACS purified populations showing Flatr expression relative to thymus Treg values. N = 6 per group. Data was normalized to RNA amount, Ppia and Rpl expression. Naïve CD4⁺ T cells (CD4⁺CD8⁻ CD62L⁺ CD44⁻); Treg, regulatory T cells (CD4⁺Foxp3⁺); DP, double positive CD4⁺CD8⁺ thymocytes; SP8, CD4⁻CD8⁺ thymocytes; SP4, CD4⁺CD8⁻ thymocytes; B cell (CD4⁻CD8⁻CD19⁺); CD8, CD8⁺ T cell; CD4 ACT, activated CD4⁺ T cells (CD4⁺CD8⁻CD44hiCD62Llo); Nrp1, Neuropilin.

Figure 3

Characterization of Flatr-expressing Treg. (A) RNA Primeflow histogram showing expression of Flatr in different immune subsets, pooled from three mice. (B) Expression of activation markers in splenic Flatr^low Treg and Flatr^high Treg (n = 4). RT-qPCR analysis showing Foxp3 (C) and Flatr (D) in cultured naïve T cells (CD4⁺ CD62L⁺ CD44⁻) from spleen and lymph nodes of wildtype mice activated in the presence of αCD3 and αCD28, with and without TGFβ at the indicated time points (n = 3–8). Normalized to Ppia and Rpl expression levels. All data are means ± SEM. *p < 0.05, **P < 0.01.

Figure 4

Generation of Flatr deficient mice. (A) Schematic depiction of Flatr locus on Chr13 and targeting strategy for exon 1 KO mice with CrispR-Cas9 mediated homologous recombination deleting 494bp containing the majority of exon 1 and ca. 230bp upstream region. HR, homologous region (yellow). Putative promoter region indicated with gray arrow. CrispR binding sites are marked in red. (B) Schematic depiction of the Flatr locus on Chr13 and targeting strategy for exon 2 with CrispR-Cas9 mediated donor-free deletion of 768bp containing exon 2, 4bp upstream and 131 downstream sequence. Promoter region indicated with gray arrow. CrispR binding sites are marked in red. (C) RT-qPCR analysis showing Flatr Foxp3 expression levels in WT, Exon1KO and Exon2KO FACS-purified Treg cells relative to WT levels (n = 6). Normalized to Ppia and Rpl expression levels.

Figure 5

Flatr deficient T cells show normal thymic Treg induction. (A) Percentage of CD4 thymic SP Foxp3⁺ Treg cells from wild-type (WT), Exon 1 KO and Exon 2 KO mice (n = 6–9). (B) Percentage of CD4⁺ and CD8⁺ cells within total lymphocytes from lymph nodes (LN) of WT, Exon 1 KO, and Exon 2 KO mice (n = 6–9). (C) Percentage of CD4⁺ Foxp3⁺ Treg cells in LN from WT, Exon 1 KO, and Exon 2 KO mice (n = 8–12). (D) Percentage of Nrp1⁺ Foxp3⁺ Treg cells in LN from WT, Exon 1 KO, and Exon 2 KO mice (n = 6–9). (E) CD25 (MFI) in the CD25⁺ Treg cell population from LN of WT, Exon 1 KO, and Exon 2 KO mice (n = 6–9). (F) RT-qPCR analysis showing Foxp3 expression levels in splenocytes from WT, Exon 1 KO, and Exon 2 KO mice relative to WT levels (n = 3). Normalized to Ppia and Rpl expression levels. (G) Foxp3 mean fluorescence intensity (MFI) in the Foxp3⁺ Treg cell population from LN of WT, Exon 1 KO, and Exon 2 KO mice (n = 4–9).

Figure 6

Flatr deficient T cells show delayed Treg induction ex vivo and in vivo. (A) Cultured naïve T cells (CD4⁺ CD62L⁺ CD44⁻) from spleen and lymph nodes of wildtype mice and Exon1 KO mice were activated in the presence of IL-2, αCD3, and αCD28, with and without TGFβ at the indicated time points (n = 3, representative of 8 experiments). *P < 0.05, **P < 0.01 for WT vs Exon1 KO at concentration of 0ng of TGFβ; #P < 0.05, ##P < 0.01 for WT vs. Exon1 KO at concentration of 2.5 ng of TGFβ; §P < 0.05, §§P < 0.01 for WT vs. Exon1 KO at concentration of 5ng of TGFβ. All data are means ± SEM. (B) Experimental design for oral antigen-induced Treg generation. 10⁶ naïve OT-II cells were transferred to a CD45.1 host. Mice were gavaged with OVA until analysis. (C) Frequency of Foxp3⁺ cells among transferred OT-II T cells (n = 4). (D) Foxp3 mRNA levels in OT-II iTreg (n = 4). (E) Flatr expression in OT-II iTreg (n = 4). (F) Experimental design for Treg induction through homeostatic expansion. Naïve CD4 T cells from both wildtype (CD45.1) and Exon1-deficient mice (CD45.2) were transferred into a Rag-deficient host, and assessed after homeostatic expansion. (G) Flow cytometric quantification of Treg number in host mice, within the wildtype (CD45.1) and Exon1KO (CD45.2) compartments. (H) CD45.1 and CD45.2 CD4 Teff and Treg cells were sorted from homeostatic expansion mice, with RT-qPCR analysis of Foxp3 and (I) Flatr (n = 4–8). Expression was normalized to Ppia and Rpl expression levels. All data are means ± SEM.

Figure 7

Flatr deficient Tregs retain normal function. (A) in vitro suppression assay data for the proliferation of naïve T cells (CD4⁺ CD62L⁺ CD44⁻) following the addition of Foxp3⁺ Tregs purified from the spleen and lymph nodes from WT and KO mice. Proliferation at different naive/Treg ratios shown (n = 6). The p-value in 1:1 ratio refers to Exon1 KO versus WT. (B) Percentage of lymph node Foxp3⁺ Treg cells expressing CTLA4. Data collected by flow cytometry from wildtype, Exon 1 KO and Exon 2 KO mice (n = 6–9). (C–F) IFNγ, IL-2, IL-10, and IL-17 production by CD4⁺ and CD8⁺ T cells from WT, Exon 1 KO and Exon 2 KO mice (n = 3–6). All data are means ± SEM. *p < 0.05.