MOESM3 of Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells

Additional file 3: Figure S1. Experimental scheme of Foxp3 stability assay. Upper, naïve CD4+ T cells (CD4+CD62L+hCD2-) were MACS sorted and cultured under iTreg skewing conditions, and on day 2, dCas9-fusion protein and gRNA were transduced with polybrene. The next day, iTregs were harvested and further cultured under iTreg or inflammatory cytokine conditions for 2 days. Foxp3 expression (hCD2) was analyzed by flow cytometry. Lower, representative Foxp3 expression. Flow cytometry plots show expression of Foxp3 (endogenous) and Foxp3(hCD2, surface indicator) in primary T cells from Foxp3-hCD52-hCD2 KI mice. Figure S2. dCas9-TET1CD-mediated Foxp3 stabilization. (A) Histogram of Foxp3(hCD2) in dCas9-TET1CD (GFP/DsRed(+/-)) and dCas9-TET1CD with #C2-7 (GFP/DsRed(+/+)) cells under inflammatory conditions. Related to Figure  3b. (B) Foxp3 mRNA expression same as in Figure 3b. Data are pooled from three independent experiments and represent the means ± SDs. Figure S3. TGF-β signal enhanced effectiveness of dCas9-p300CD-mediated Foxp3 induction. Foxp3 expression induced by low-dose TGF-β in the presence of LY2157299 or anti-TGF-β was monitored by Foxp3(hCD2) MFI. Figure S4. dCas9-p300CD and gRNA co-transduced iTregs. (A) Sorting strategy and purification. (B) Suppression assay of iTregs comparing dCas9-p300CD and #P-4 with dCas9-p300CD catalytic mutant

MOESM2 of Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells

Additional file 2: Table S1. Off-target analysis of selected gRNAs

Prevention of graft rejection by Tregs.

<p><b>A,</b> Cardiac graft survival in mice treated with antigen-specific iTregs (CBA background) induced by the indirect pathway and direct pathway. Tregs (1×10⁶ cells) were injected intravenously one day before transplantation of the C57BL/6 heart into CBA recipients. BALB/c to CBA cardiac transplantation was performed after injection of indirect-iTregs (CBA) against the K^b peptide. (The Log-rank test was used; <i>P</i> values are compared between the indirect pathway and other pathways; no treatment, <i>P</i><0.001; anti-CD3 Ab/anti-CD28 Ab, <i>P</i><0.001; BALB/c vs. CBA, <i>P</i> = 0.0013; direct pathway, <i>P</i><0.001. Direct-iTreg treatment vs. no treatment, <i>P</i><0.001) <b>B,</b> HE staining of harvest cardiac grafts indicated days after transplantation. The bar of the right lower panels was 10 µm. Magnification; 200×.</p

STAT3 expression by myeloid cells is detrimental for the T- cell-mediated control of infection with <i>Mycobacterium tuberculosis</i>

<div><p>STAT3 is a master regulator of the immune responses. Here we show that <i>M</i>. <i>tuberculosis</i>-infected <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> mice, defective for STAT3 in myeloid cells, contained lower bacterial load in lungs and spleens, reduced granuloma extension but higher levels of pulmonary neutrophils. STAT3-deficient macrophages showed no improved control of intracellular mycobacterial growth. Instead, protection associated to elevated ability of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> antigen-presenting cells (APCs) to release IL-6 and IL-23 and to stimulate IL-17 secretion by mycobacteria-specific T cells. The increased IL-17 secretion accounted for the improved control of infection since neutralization of IL-17 receptor A in <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> mice hampered bacterial control. APCs lacking SOCS3, which inhibits STAT3 activation via several cytokine receptors, were poor inducers of priming and of the IL-17 production by mycobacteria-specific T cells. In agreement, <i>socs3</i>^<i>fl/fl</i> <i>cd11c cre</i> mice deficient of SOCS3 in DCs showed increased susceptibility to <i>M</i>. <i>tuberculosis</i> infection. While STAT3 in APCs hampered IL-17 responses, STAT3 in mycobacteria-specific T cells was critical for IL-17 secretion, while SOCS3 in T cells impeded IL-17 secretion. Altogether, STAT3 signalling in myeloid cells is deleterious in the control of infection with <i>M</i>. <i>tuberculosis</i>.</p></div

SOCS3 and STAT3 in antigen-specific T cells are important regulators of IL-17 and IFN-γ secretion.

<p><i>Stat</i>3^<i>fl/fl</i><i>lck cre p25-tg</i> T cells (A, B), <i>socs3</i>^<i>fl/fl</i> <i>lck cre p25-tg</i> T cells (C, D) or control <i>p25-tg</i> T cells were incubated for 3 days with either BCG, peptide 25 and LPS-stimulated or untreated BMDCs. The mean concentration of IL-17 (A, C) and IFN-γ (B, D) ± SEM in supernatants from triplicate cultures is shown (**p<0.01 and ***p<0.001 Student’s t test). <i>Graphical summary</i> (E): Mice deficient in STAT3 in myeloid cells show increased resistance while <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> mice displaying augmented STAT3 activation had impaired resistance against infection with <i>M</i>. <i>tuberculosis</i>. The differential control of infection in excess or deficiency of STAT3 is not due to the intrinsic regulation of bacterial control by macrophages but rather to differences in the ability of DCs to regulate the differentiation of specific T-cells. <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs release higher levels of IL-6 and IL-23 after stimulation with mycobacterial or other TLR agonists, while secretion of these cytokines is reduced in <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs. <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs show improved ability to trigger IL-17 release by mycobacteria-specific T cells while the opposite is observed when using <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> APCs. The IL-17 secretion by T cells is also controlled via gp130R signalling, indicating an autocrine or paracrine loop by IL-6 family cytokines. The increased resistance to <i>M</i>. <i>tuberculosis</i> infection of <i>stat3</i>^<i>fl/fl</i> lysm cre mice was IL-17 dependent. <i>Socs3</i>^<i>fl/fl</i> <i>lysm cr</i>e (but not <i>stat3</i>^<i>fl/fl</i> <i>lysm cre)</i> DCs improved capacity of mycobacteria-specific T cells priming <i>in vivo</i>.</p

Stat3 deficient and control BMM show similar control of the intracellular growth of <i>M</i>. <i>tuberculosis</i>.

<p>The levels of <i>tnf</i> mRNA in the lungs from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice at the indicated time points after infection with <i>M</i>. <i>tuberculosis</i> (A), and in <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM incubated with <i>M</i>. <i>tuberculosis</i> (C) or BCG (D) were determined by real time PCR. The mean fold increase of mRNA level ± SEM in 8 mice per group (A) or in triplicate independent cultures per condition compared to non-infected cultures (C, D) of one of two independent experiments is depicted (*p<0.05, **p<0.01, ***p<0.001 Student t test). The mean concentration of TNF in the supernatants of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM at different times after <i>M</i>. <i>tuberculosis</i> infection were measured by ELISA (B). The mean % of infected BMM (E), the bacteria number per BMM (F) and the bacteria per infected BMM (G) ± SEM at 0 and 3 days after infection of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM were determined after staining with auramin-rhodamin T for mycobacterial and DAPI for nuclei (E-G). One out of 2 independent experiments performed is depicted. Bacterial CFU were determined in <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMM after infection with BCG (H) or <i>M</i>. <i>tuberculosis</i> (I) at a MOI of 5:1. The mean CFU ± SEM from triplicate cell cultures is shown. Three independent experiments for each panel were performed.</p

Expansion of Tregs by BMDCs <i>in vitro</i>.

<p><b>A, B, </b><i>In vitro</i> antigen-specific expansion of iTregs via the indirect pathway. Naïve CD4⁺ T cells from CBA mice were cultured with BMDCs (CBA) in the presence of the K^b peptide for 7 days at the indicated conditions. The Treg fraction was determined as CD3⁺CD4⁺ CD25⁺Foxp3⁺ (***<i>P</i><0.001, Bonferroni test) <b>C, D,</b> The-time course of Treg fraction (C) and number (D) in the <i>in vitro</i> culture with BMDCs in the presence of 10 µg/ml K^b peptide, 10 ng/ml IL-2, 2 ng/ml TGF-β, 5 µg/ml anti- IFN-γ and anti-IL-4 antibodies, and 20 nM ATRA. <b>E,</b> The cell surface marker and purity of the Tregs before and after cell sorting, on day 8 after culture via the indirect and direct pathway. Tregs induced via the direct pathway were cultured with C57BL/6 BMDCs and 10 ng/ml IL-2, 2 ng/ml TGF-β, 10 µg/ml anti- IFN-γ and 5 µg/ml anti-IL-4 antibodies, and 20 nM ATRA. The cells were stained with the indicated antibodies and analyzed with FACS.</p

STAT3 and SOCS3 regulate the secretion of IL-17 by antigen-specific T cells.

<p>Total RNA was extracted from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC cultures 24 h after <i>M</i>. <i>tuberculosis</i> infection. <i>Il6</i> and <i>il23p19</i> mRNA were measured by real time PCR and normalized to the <i>hprt</i> mRNA levels in the same samples. The mean and <i>il6</i> (A) <i>il-23p19</i> (B) mRNA fold increase ± SEM levels in triplicate independent cultures are depicted (*p<0.05, **p<0.01 and ***p<0.001 Student’s t test). The mean fold increase of <i>il6</i> (C) and <i>il23p19</i> (D) ± SEM were measured by real-time PCR in triplicate cultures of stat3^<i>fl/fl</i> <i>lysm</i> cre and <i>stat3</i>^<i>fl/fl</i> BMDCs 24 h after stimulation with either LPS, CpG or Pam3K (*p<0.05 and **p<0.01 Student’s t test). <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC (E-H) or BMM (H) were stimulated with either BCG (E), heat killed BCG (F), <i>M</i>. <i>tuberculosis</i> (G), or with LPS and the peptide 25 of Ag85b (H) and incubated 6 h after with <i>p25-tg</i> CD4+ naïve T cells (at a ratio of 4:1 BMDC). The concentration of IL-17 in the culture supernatants was measured by ELISA 72h after co-incubation. The mean IL-17 ng/ ml ± SEM from triplicate cultures is depicted (* p<0.05; **p<0.01 and ***p<0.001 Student’s t test). Total RNA was extracted from <i>socs3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>socs3</i>^<i>fl/fl</i> BMDC cultures at different times after infection with mycobacteria. The mean <i>Il6</i> (I) and <i>IL23p19</i> (J) mRNA levels ± SEM levels determined by real time PCR are depicted (**p<0.01 Student’s t test). The concentration of IL-17 was measured 72h supernatants of co cultures of <i>p25-tg</i> naïve T cells incubated with either BCG (K), <i>M</i>. <i>tuberculosis</i> (L) or pept25 and LPS (M)-stimulated socs3^<i>fl/fl</i> <i>lysm cre</i> and socs3^<i>fl/fl</i> BMDCs. The mean IL-17 ng/ ml ± SEM from triplicate independent cultures were determined by ELISA (*p<0.05; **p<0.01 and ***p<0.001 Student’s t test). IL-17 was measured in 72 h culture supernatants from control or BCG-infected <i>gp130</i>^<i>F/F</i> BMDC and <i>p25-tg</i> T cells. The mean IL-17 ng/ ml ± SEM from triplicate cultures from infected or control BMDCs is shown in (N). Differences are significant at ***p<0.001 Student’s t test. The mean fold increase of <i>socs3</i> transcript ± SEM in total RNA from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> BMDC at different time points after <i>M</i>. <i>tuberculosis</i> infection as compared to uninfected controls were measured by real time PCR are depicted (O).</p

Myeloid cell expression of STAT3 hamper IL-17 secretion by T cells during M. tuberculosis infection.

<p>The frequency of IL-17-secreting PPD and PMA and ionomycin (P.I) -stimulated CD4+ pulmonary T cells from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 4 (A) and 8 (B) weeks after infection with <i>M</i>. <i>tuberculosis</i> was measured by FACS. A representative graph plot from PPD stimulated lungs at 4 w after infection (C) and the mean percentage of IL-17-secreting CD4+ cells ± SEM (A, B) are displayed (n = 6 per group, *p<0.05, **p<0.01 and ***p<0.001 Mann Whitney U test). The mean fold increase of <i>il17a</i> (D), <i>il22</i> (E), <i>cxcxl5</i> (F), <i>il23p19</i> (G) and <i>il6</i>(H) mRNA ± SEM was measured by real time PCR in the total RNA from lungs of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice at different time points after <i>M</i>. <i>tuberculosis</i> infection (n = 8 per group **p<0.01 Student’s t test). The mean frequency of PPD (I) and PMA and ionomycin (J) -stimulated CD4+ pulmonary T cells from <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 8 weeks after infection with <i>M</i>. <i>tuberculosis</i> co-secreting or not IFN-γ was measured by FACS (n = 4 per group, *p<0.05, **p<0.01 and ***p<0.001 Mann Whitney U test). A representative graph plot from CD3+CD4+ gated PPD and P.I. stimulated IFN-γ and / or IL-17+ lung cells (K) is depicted. The frequency of both IL-17 and IFN-γ and only IL-17+ secreting cells from <i>stat3</i>^<i>fl/fl</i><i>lysm cre</i> is higher as compared to <i>stat3</i>^<i>fl/fl</i> controls; the frequency of IL-17+/IFN-γ- is higher than IL-17+/IFN-γ+ cells. This was determined after either PPD or PMA/ ionomycin stimulation (Two-way ANOVA *p<0.05 and *** p<0.0001). <i>Stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> littermate controls were treated i.p. 1 day before and once per week after aerosol infection with <i>M</i>. <i>tuberculosis</i> with 500 μg anti-IL-17RA M751or left untreated. Mice were sacrificed 4 weeks after the infection and colony forming units (CFU) per lung (L) and spleen (M) were assessed. The CFU per organ of individual mice and the median per group at the indicated time points after infection are depicted. Differences in CFU are significant (*p<0.05, **p<0.01 Mann Whitney U test). The mean fold increase of <i>mpo</i> mRNA ± SEM was measured by real time PCR in lysates from lungs of <i>stat3</i>^<i>fl/fl</i> <i>lysm cre</i> and <i>stat3</i>^<i>fl/fl</i> mice 4 weeks after infection with <i>M</i>. <i>tuberculosis</i> treated or not with anti-IL-17RA as described above (n≥ 4 per group, *p<0.05 Student’s t test) (N).</p

Tregs features in the cardiac grafts on day 7 after transplantation(n = 3–5).

<p><b>A,</b> Proportion and cell number of Tregs in each graft. Mononuclear cells were separated from the graft by collagenase treatment and Percoll gradient centrifugation. After staining with CD3, CD4, CD25 and Foxp3 antibodies, cells were analyzed with FACS. In the lower panel, the number of infiltrated Tregs per graft is shown (n = 5). <b>B,</b> Mononuclear cells in the grafts were stained with anti-CTLA4 (left) and anti-IL-10 (left) antibodies after PMA, ionomycin, and brefeldin treatment, and analyzed with FACS (n = 3–5). The proportion of CTLA4- and IL-10-producing cells in the CD4⁺ T cell population in the grafts is shown. <b>C,</b> The cell number of IFN-γ and IL-6-producing cells. The mononuclear cells infiltrated into the grafts were stained with anti- IFN-γ and anti-IL-6 antibodies and analyzed with FACS (n = 3–5). (*** <i>P</i><0.01, ** <i>P</i><0.03, * <i>P</i><0.05, Tukey's Multiple Comparison Test).</p