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Received 3 Nov 2014 | Accepted 28 Jan 2015 | Published 6 Mar 2015

Alvin Pratama1, Monika Srivastava1, Naomi J. Williams1, Ilenia Papa1, Sau K. Lee1, Xuyen T. Dinh2, Andreas Hutloff3, Margaret A. Jordan2, Jimmy L. Zhao4, Rafael Casellas5, Vicki Athanasopoulos1 & Carola G. Vinuesa1

Tight control of T follicular helper (Tfh) cells is required for optimal maturation of the germinal centre (GC) response. The molecular mechanisms controlling Tfh-cell differentiation remain incompletely understood. Here we show that microRNA-146a (miR-146a) is highly expressed in Tfh cells and peak miR-146a expression marks the decline of the Tfh response after immunization. Loss of miR-146a causes cell-intrinsic accumulation of Tfh and GC B cells. MiR-146a represses several Tfh-cell-expressed messenger RNAs, and of these, ICOS is the most strongly cell autonomously upregulated target in miR-146a-decient T cells. In addition, miR-146a deciency leads to increased ICOSL expression on GC B cells and antigen-presenting cells. Partial blockade of ICOS signalling, either by injections of low dose of ICOSL blocking antibody or by halving the gene dose of Icos in miR-146a-decient T cells, prevents the Tfh and GC B-cell accumulation. Collectively, miR-146a emerges as a post-transcriptional brake to limit Tfh cells and GC responses.

1 Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Building 131, Garran Road, Canberra, Australian Capital Territory 0200, Australia. 2 Comparative Genomics Centre, James Cook University, Townsville, Queensland 4811, Australia. 3 Chronic Immune Reactions Group, German Rheumatism Research Centre Berlin (DRFZ), a Leibniz Institute, 10117 Berlin, Germany. 4 Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA. 5 Genomics and Immunity Section, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. Correspondence and requests for materials should be addressed to C.G.V. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms7436 OPEN

MicroRNA-146a regulates ICOSICOSL signalling to limit accumulation of T follicular helper cells and germinal centres

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7436

Tfollicular helper (Tfh) cells provide essential survival and selection signals to germinal centre (GC) B cells that are important for long-lived protective antibody responses1,2.

Increasing evidence suggests that restricting Tfh-cell numbers in GCs is crucial for optimal GC B-cell selection35. B cells expressing the highest afnity receptors after somatic hypermutation can capture more antigens and therefore have a competitive advantage in establishing sustained interactions and eliciting survival signals from Tfh cells5. Studies of autoimmune mouse models69 and human patients1014 suggest that excessive Tfh cells may contribute to the pathogenesis of antibody-mediated autoimmune diseases, potentially by allowing survival and differentiation of self-reactive B cells. While multiple signals are now recognized to be important for Tfh-cell formation and migration3, relatively little is known about the mechanisms that limit Tfh-cell numbers to achieve optimal selection of high afnity B-cell clones. Cell-extrinsic mechanisms such as the actions of T follicular regulatory (Tfr)1517 and follicular CD8

T cells18 have been reported, but to date, only Roquin is shown to act in a T cell-autonomous manner to prevent spontaneous accumulation of Tfh cells19.

MicroRNA-146a (miR-146a) has recently emerged as a key post-transcriptional regulator in hematopoietic cells. MiR-146a represses TRAF6 and IRAK1 in myeloid cells20 and T cells21 to control their proliferation and NF-kB activation in response to

Toll-like receptor and TCR signalling, respectively. Deciency of miR-146a leads to excessive production of IL-6 and TNF, myeloproliferation, chronic inammation and a decline in the number and quality of hematopoietic stem cells20,22,23. In the absence of miR-146a, regulatory T (Treg) cells also lose their suppressive ability due to STAT1 overexpression driving increased IFN-g secretion24. Not surprisingly, dysregulated expression of miR-146a has also been found to correlate with increased incidence of autoimmune diseases, such as lupus2528 and rheumatoid arthritis2932.

Here we show that miR-146a profoundly represses Tfh-cell numbers: the absence of this miRNA leads to spontaneous Tfh-cell accumulation that precedes myeloid cell dysregulation and is not a consequence of Treg-cell functional deciency. This is achieved by directly repressing multiple messenger RNAs (mRNAs) targets, most prominently Icos.

ResultsMiR-146a is highly expressed in Tfh cells. To identify miRNAs that might regulate Tfh-cell development and/or function, we performed a genome-wide expression analysis to identify miRNAs that are highly expressed in human Tfh cells. Compared with nave (CD44low CD25 ) CD4 T cells, miR-146a was the most differentially expressed miRNA (8.2-fold) in Tfh cells (Fig. 1a) among those that are highly expressed in T cells. Real-time PCRs on T-cell subsets from human tonsils revealed that Tfh cells expressed the highest amounts of miR-146a compared with all other effector (CD44high) subsets (Fig. 1b). Consistent with these results, a deep-sequencing survey of the miRNome in mouse tissues showed the highest levels of miR-146a in Tfh and GC B cells (Fig. 1c and ref. 33). Lower amounts of miR-146a were also present in activated B cells and thymic-derived Tregs, while non-haematopoietic tissues displayed little or no miR-146a expression (Fig. 1c).

MiR-146a expression peaks at the late stage of GC responses. To study the kinetics of miR-146a expression during a T cell-dependent immune response, we adoptively transferred 5 105

nave CD4 T cells from Ly5b OT-II TCR-transgenic mice into Ly5a wild-type C57BL/6 recipient mice, which were then

immunized with ovalbumin (OVA) precipitated in alum and killed at different time points (Fig. 1d). The proportion of Tfh cells among donor cells peaked at day 7 post immunization (Fig. 1e) and started to contract by day 10, coinciding with the peak of miR-146a expression in transferred cells (Fig. 1e). This suggests a possible role for miR-146a in downsizing the Tfh-cell population.

Cell-autonomous control of Tfh cells by miR-146a. To test whether miR-146a plays a role in the control of Tfh-cell numbers, we studied Tfh-cell development and function in 12- to 16-week-old mice lacking miR-146a. Analysis of total follicular T cells, gated as CXCR5high PD-1high (Fig. 2a,b) or BCL-6high PD-1high (Fig. 2c,d) cells, showed they were expanded in both percentage and total number in unimmunized Mir146a / mice compared with wild-type littermates. This expansion was not selective to either Tfh (CXCR5high PD-1high Foxp3 ) or Tfr (CXCR5high

PD-1high Foxp3 ) cells as both subsets increased in parallel (Fig. 2e). This effect of miR-146a deciency seemed to affect Tfh cells more severely than the other effector CD4 T cell subsets: compared with the 5-fold increase in Tfh cells, no statistically signicant increases were observed in either Th2 (IL-4 ) cells or

Th17 (IL-17 ) cells, while Th1 (IFN-g ) cells were increased by1.4-fold and Tregs (Foxp3 ), as previously reported24, were expanded by 1.5-fold (Fig. 2f). Total GC B cells were also increased by about 20-fold in Mir146a / mice compared with wild-type littermates (Fig. 2g,h). The spontaneous accumulation of Tfh and GC B cells became more severe with age (Supplementary Fig. 1).

To test whether the accumulation of Tfh cells in Mir146a / mice was due to T-cell intrinsic factors, we set up mixed bone marrow chimeras by transferring equal amounts of either Ly5a .Mir146a / : Ly5b .Mir146a / (WT:WT) or Ly5a .

Mir146a / : Ly5b .Mir146a / (WT:KO) bone marrow cells into sub-lethally irradiated Rag1 / recipients. Four months after reconstitution, the spleens were harvested and the percentage of total CD4 T cells, effector cells, Tfh cells, total

B220 cells and GC B cells derived from the Ly5a or Ly5b donor marrow in each mouse was determined (Fig. 3ad). There was a higher percentage of total follicular T cells in the WT:KO chimeras compared with the WT:WT chimeras (Fig. 3a). This was due to cell-autonomous expansions of Tfh and Tfr cells derived from Ly5b .Mir146a / marrow (Fig. 3b). In each mouse, there was a two to fourfold increase in Ly5b Tfh cells compared with Ly5a Tfh cells, whereas this was not observed in the control WT:WT chimeras, in which the percentages of Ly5a and Ly5b Tfh cells were comparable (Fig. 3b). Importantly, the percentages of non-follicular effector CD4 cells were comparable between Ly5a and Ly5b cells in both WT:WT and

WT:KO mixed chimeras. Consistent with previous reports24, there was a cell-autonomous expansion of non-follicular Tregs derived from Ly5b .Mir146a / marrow in the WT:KO chimeras (Fig. 3b).

We also observed a statistically signicant increase in the percentage of GC B cells derived from Ly5b .Mir146a /

marrow compared with those derived from Ly5a .Mir146a / bone marrow (Fig. 3c,d), suggesting that miR-146a also acts cell autonomously in GC B cells. Intriguingly, despite the signicant increase of total follicular T cells in the WT:KO chimeras (Fig. 3a), we only observed expansion of the Ly5b .Mir146a /

GC B cells, while the percentage of the neighbouring Ly5a .Mir146a / GC B cells was comparable to that in the WT:WT chimeras (Fig. 3d). This could indicate that GC expansion requires the concerted actions of miR-146a in T cells and B cells, perhaps through the regulation of a receptorligand pair in each

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7436 ARTICLE

Naive CD4+cells

(Log 2mean expression)

3,000

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10 ***

***

Naive CD4 T

Non-Tfh effector

Treg

miR-146a

4 5

hsa-miR-146a

2 2 1

Tfh cells (Log2 mean expression)

0 1 2 3

1.5-fold

Tfh

Tfr

mmu-miR-146a (tags per million)

ProB

0 Immature

PreB

LPS+--dextran B

LPS+IL-4 B

Mature

MZ GC

PCs

Mast cells

HSCs

Basophils

Neutrophils

Macrophages

DCs

CD4+ CD8+

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CD8

CD8 ConA

Th1

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iTreg

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ESCs

MEFs

Heart

Brain

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Ovaries

Tfh cells frequency

miR-146a expression

30 20 10

5 105 sorted

Nave CD4+ T cells

+ OVA-alum

Recipients:

Ly5a+ WT B6 mice

d0 d3 d5 d7 d10

(% donor CD4+cells)

40 150

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mmu-miR-146a

Tfh cells

(on donor CD4+cells)

Donors:

Ly5b+ OT-II mice

d12

Cell transfer

and immunization

Tfh and miR-146a expression

analysis on donor Ly5b+ cells

0 3 5 7 10 12

Days post immunization

Figure 1 | MiR-146a is highly expressed in Tfh cells. (a) Microarray analysis of miRNA expression in nave CD4 T and Tfh cells. The scatter plot shows the log2 of averaged (n 3) background-subtracted expression of all Agilent Human miRNA Microarray (V1) Kit 8 15K probes by Tfh cells on the x-axis

compared with nave CD4 T cells on the y-axis. Each dot represents one miRNA probe. The dotted lines are drawn at 1.5-fold difference in expression. (b) Expression of miR-146a, normalized to U6, measured by real-time PCR from human tonsillar T-cell subsets. Cell sorting strategy is described in the

Methods. The heights of the bars represent the mean, and the error bars represent the s.d. of three technical replicates. Data are representative of three independent experiments. Statistical signicance was determined using unpaired Students t-test. ***Po0.001, ****Po0.0001. (c) Bar graphs showing the expression of miR-146a in mouse immune cell subsets and non-hematopoietic tissues as described in Kuchen et al.33. In brief, small RNAs from various mouse tissues were ligated to adapters using Illuminas protocol. These were then reverse transcribed, amplied by PCR and sequenced on a Genome Analyzer (Illumina). (d,e) Kinetics of the Tfh-cell response (lled circles) and miR-146a expression (open circles) from transferred Ly5b OT-II cells analyzed at different time points according to the strategy shown in d. Three recipient mice were killed at each time point. Percentage of Tfh cells among donor Ly5b cells was measured using ow cytometry staining of CD4 CXCR5high PD-1high Foxp3 T cells. MiR-146a expression was measured by real-time PCR of total Ly5b CD4 Tcells and was normalized to U6 expression. Filled and open circles in e represent the mean, and error bars represent the s.d. of biological replicates (for Tfh-cell percentage) or technical replicates (for miR-146a expression).

cell type. Collectively, these results suggest that miR-146a acts in T cells and B cells to prevent Tfh and GC B-cell accumulation.

MiR-146a deciency in T cells initiates Tfh-cell expansion. We next investigated whether accumulation of Tfh cells could occur independently of neighbouring Mir146a / B cells and myeloid cells in the same mice. To this end, we transferred puried nave

CD4 T cells from Mir146a / or Mir146a / OT-II TCR-transgenic mice into Cd28 / recipients, which were then immunized with OVA precipitated in alum and killed 7 days later. There was a statistically signicant increase in the percentage of OVA-specic Vb5 Tfh cells and in the percentage of total GC B cells in mice that received Mir146a / OT-II cells, although the magnitude of the effect was smaller than that

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7436

+/+ /

10 **

8 6 4

105

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CXCR5hi PD-1hi cells (103)

1,000 **

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BCL-6hi PD-1hi

(% of CD4+ cells)(% of CD4+ cells)

Mir146a : +/+ /

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CXCR5hi PD-1hi

PD-1

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1,000

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+/+ /

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Tfr (% of CD4+ cells)

PD-1

# Cells

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CXCR5

Foxp3

Mir146a :

0 +/+

/ +/+ /

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IFN-+ cells (% CD4+ cells)

* 0.20 20 3

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** *

0.200.150.100.050.00

+/+ / +/+ /

IL-17+ cells (% CD4+ cells)

0.15

0.10

0.05

0.00

Foxp3+ cells (% CD4+ cells)

Tfh cells (% CD4+ cells)

Mir146a :

+/+ /

IL-4+ cells (% CD4+ cells)

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+/+ /

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GC B (% of B220+ cells)

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1 +/+ /

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0.089 1.87

GL-7

102

103

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105

102

103

104

105

Fas

Mir146a :

0 +/+ /

Figure 2 | Loss of miR-146a causes spontaneous accumulation of Tfh and GC B cells. (ad) Flow cytometric contour plots and dot plots showing percentages and absolute cell number of follicular T cells gated as CXCR5high PD-1high cells (a,b) or BCL-6high PD-1high cells (c,d). (e) Gating strategy for Tfh (CXCR5high PD-1high Foxp3 ) and Tfr (CXCR5high PD-1high Foxp3 ) cells and dot plots showing their percentages. (f) Percentages of Th1 (IFN-g),

Th2 (IL-4 ), Th17 (IL-17 ), Treg (Foxp3 ) and Tfh (CXCR5high PD-1high Foxp3 ) cells among total CD4 cells. (g,h) Flow cytometric contour plots and dot plots showing percentages and absolute cell numbers of GC B cells. Contour plots shown in a,c,e are gated on CD4 cells, those in g are gated on

B220 cells. Unimmunized miR-146a-decient ( / ) and -sufcient ( / ) mice were analyzed. Each symbol represents one mouse and the

horizontal bars represent the median values. Data are representative of at least three independent experiments. Statistical signicance was determined using MannWhitney U-test. NS, not signicant, *Po0.05, **Po0.01.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7436 ARTICLE

50% Mir146a+/+:

50% Mir146a+/+

50% Mir146a+/+:

50% Mir146a /

10 NS

***

105

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Tfh cells (% CD4+ cells)

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Ly5:

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4.59

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Ly5a

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0 40 ** ****

30 20 10

Non-follicular effector cells

(% CD4+ cells)

Non-follicular Tregs

(% CD4+ cells)

102

0 105

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30 20 10

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Ly5: a

PD-1

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0 105

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CXCR5

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50% Mir146a/

***

105

1.59

6 4 2 0

Ly5a

Tfh (% of donor OT-II cells)

1.49

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Mir146a:

Ly5: a b a b

+/+ +/+ +/+ /

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GC B cells (% of B220+ cells)GC B (% of B220+ cells)

5.97

Ly5b

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Mir146a:

+/+

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B cells Mir146a +/+

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Tfh cells (% CD4+ cells)

CD4+ T cells

Rag1/

recipients

14 days

or + or

SRBC

immunization

15 10

5 0

15 10

5 0

+/+ /

+/+

Mir146a (T cells):

Mir146a (B cells):

+/+

Figure 3 | Cell-autonomous accumulation of miR-146a-decient Tfh cells. (a) Flow cytometric contour plots showing the gating strategy for follicular T cells in 50:50 mixed bone marrow chimeras gated on all CD4 (top), Ly5a CD4 (middle) and Ly5b CD4 (bottom) cells. (b) Percentages of

Tfh, Tfr, non-follicular effector and non-follicular Tregs in the mixed chimeras. (c,d) Flow cytometric contour plots and dot plot showing the percentages of GC B cells in the mixed chimeras. Contour plots in c are gated on Ly5a B220 and Ly5b B220 cells as indicated. Connecting lines between percentages of Ly5a and Ly5b in b and d indicate that they are from the same chimeric mouse. Chimeras were analyzed 16 weeks post reconstitution.

(e) Percentages of OVA-specic (Vb5 ) Tfh cells and total GC B cells in Cd28 / recipient mice 7 days following adoptive transfer. Data are representative of three independent experiments. (f,g) Adoptive transfer of puried CD4 T cells and B cells from Mir146a/ or Mir146a / donor mice into Rag1 / recipients according to the strategy shown in f. The recipient mice were immunized with sheep red blood cells 14 days after transfer and they were killed 7 days post immunization. (g) Percentages of Tfh and GC B cells in Rag1 / recipient mice following adoptive transfer and SRBC immunization. Each symbol represents one mouse and the horizontal bars represent the median values. Statistical signicance was determined using a paired Students t-test (b,d) or MannWhitney U-test (e,g). NS, not signicant, *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001.

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7436

observed in intact Mir146a / mice (Fig. 3e), suggesting that non-T cells, including B cells and myeloid cells, enhance this effect.

In a separate experiment, four different combinations of Mir146a / or Mir146a / CD4 T cells and B cells were co-transferred into Rag1 / mice, which were immunized 14 days later with sheep red blood cells (SRBCs; Fig. 3f). Similar to the

OT-II transfer experiment, we found that in the presence of wild-type B cells and myeloid cells, miR-146a deciency in T cells was sufcient to cause Tfh-cell accumulation, although the magnitude of the effect was again more modest than that observed in intact Mir146a / mice (Fig. 3g). The presence of Mir146a /

B cells appeared to exacerbate the effect, but this did not reach statistical signicance. Similar trends were observed for the increase in GC B cells (Fig. 3g). In summary, our data show that miR-146a deciency in T cells is sufcient to initiate Tfh and GC B-cell accumulation, but T-cell-extrinsic factors contribute to exacerbate this phenotype.

Contribution of IL-6 to Tfh and GC B-cell accumulation. In view of the reported increased proliferation of myeloid cells in 6- to 8-month-old Mir146a / mice20,34, we asked whether expanded myeloid compartment could be one of the T-cell-extrinsic factors contributing to the Tfh and GC B-cell expansion. Assessments of 12- to 16-week-old mice, when the Tfh-cell accumulation is obvious (Supplementary Fig. 1), revealed no signicant accumulation of myeloid cell subsets in Mir146a /

mice (Supplementary Fig. 2a).

Myeloid cells and T cells have been reported to produce more IL-6 cytokine in 6- to 10-month-old miR-146a-null mice20,23. Since IL-6 is required for Tfh-cell formation35,36, we tested whether IL-6 production was elevated in 12- to 16-week-old Mir146a / mice. No signicant increase of IL-6 was observed in the serum of these mice (Supplementary Fig. 2b), and we also did not nd increased amount of Il6 transcripts in CD11chigh splenic dendritic cells (Supplementary Fig. 2b). Next, we used Ly5a .Mir146a / : Ly5b .Mir146a / mixed bone marrow chimeras to investigate the possibility that during the course of an immune response, IL-6 might be expressed at higher levels in Mir146a / GC B cells, Tfh cells or myeloid cells. Despite the expected cell-autonomous accumulation of Mir146a / Tfh cells in the mixed chimeric mice (Supplementary Fig. 3), no differences in Il6 mRNA expression were found between miR-146a-sufcient and miR-146a-decient cells in any of the subsets examined (Fig. 4ac). Finally, we tested the possibility that follicular dendritic cells (FDCs), which are of nonhematopoietic origin, expressed more IL-6 in the absence of miR-146a; it has been suggested that FDC-derived IL-6 is important for the late stage maintenance of Tfh cells during viral infection35. We isolated FDCs according to published protocols by gating on CD45 CD31 CD21/35 cells from

Mir146a / or Mir146a / mice. As both gp38-expressing37 and non-gp38-expressing38 FDCs have been identied, we separated these two cell subsets and measured the expression of IL-6 (Fig. 4d). Again, we did not nd evidence of increased Il6 transcripts in either gp38 or gp38 FDCs from miR-146-decient mice (Fig. 4e). Nevertheless, a complete blockade of

IL-6R using a previously reported dose of monoclonal antibody35 greatly reduced Tfh-cell accumulation in Mir146a / mice (Supplementary Fig. 4), indicating that miR-146a-driven Tfh-cell accumulation is still dependent on IL-6-mediated signalling.

Intriguingly, we observed enhanced IL-6-induced STAT3 phosphorylation in nave and effector CD4 T cells from

Mir146a / mice (Fig. 4f). Lack of increased STAT1 phosphor-ylation in parallel assays (Supplementary Fig. 5) argued against

STAT1 overexpression being the cause of this enhanced sensitivity to IL-6. Collectively, these results suggest that increased IL-6 expression is probably not the main cause of the Tfh-cell accumulation in miR-146a-decient mice; rather, the increased sensitivity to IL-6 signalling is a more likely contributor to the phenotype.

MiR-146a represses multiple Tfh-cell mRNAs. To uncover mRNA targets of miR-146a in T cells, we performed a microarray analysis on sorted nave CD4 T cells from Mir146a / and

Mir146a / mice. A number of differentially expressed genes were identied and for further validation, we selected putative targets based on meeting two of the following three criteria:(1) upregulated mRNA expression in Mir146a / T cells assessed by global gene expression proling, (2) known roles in

Tfh and/or GC biology and (3) the presence of target sites for miR-146a seed region within their 30 untranslated region (UTR).

The selected mRNA targets were Stat1, Cxcr4, Notch1, Slamf1, Slamf6, Cd84, Icos, Il6st and Il21r (putative miR-146a binding sites are shown in Supplementary Fig. 6; previously validated target sites in Cxcr4, Notch1 and Stat1 are not shown).

Next we assessed by ow cytometry whether the proteins encoded by these putative RNA targets were upregulated in miR-146a-decient T cells. STAT1, a validated target of miR-146a in Tregs24 and an important signalling intermediate and transcription factor downstream of IL-6, IL-12, IL-21 and IL-27, was upregulated in Mir146a / nave CD4 , Tfh cells (Fig. 5a) and, as shown previously24, in Tregs (Supplementary

Fig. 7). The validated miR-146a targets CXCR4 (ref. 39), known to regulate dark zone versus light zone partitioning in GCs5,40,41, and Notch1 (ref. 42), shown to promote Tfh-cell differentiation43, were both upregulated in Mir146a / effector CD4 , Tfh cells (Fig. 5a) and in Tregs (Supplementary Fig. 7). ICOS is required for providing costimulatory signals for optimal Tfh and GC B-cell differentiation4447 and for persistent directional motility and follicular recruitment of activated CD4 T cells48 and failed Icos mRNA repression is associated with accumulation of Tfh cells in lupus-prone Roquin-1/2 mutant mice6,19,49. We observed ICOS overexpression in Mir146a / nave, effector CD4, Tfh cells (Fig. 5a) and Tregs (Supplementary Fig. 7). Members of the

SLAM family of receptors (SLAMF1, SLAMF6 and CD84) mediate prolonged interactions between activated antigen-specic T and B cells and signal via the recruitment of SAP to promote terminal Tfh-cell differentiation50. SLAMF1, SLAMF6 and CD84 were upregulated in Mir146a / nave and effector

CD4 T cells compared with their wild-type counterparts (Fig. 5a). CD84 was upregulated in Mir146a / Tfh cells (Fig. 5a), and SLAMF1 and CD84 were also upregulated in

Mir146a / Tregs (Supplementary Fig. 7). IL-21R and IL6ST are subunits of receptors for the Tfh-cell-inducing cytokines

IL-21 and IL-6, respectively. IL-21R was upregulated in Mir146a / nave (Fig. 5a) and Tregs (Supplementary Fig. 7), while IL6ST was upregulated in Mir146a / nave and effector

CD4 T cells (Fig. 5a), but not in Tfh cells (Fig. 5a) or Tregs (Supplementary Fig. 7).

The putative targets that had not been previously validated (Slamf1, Slamf6, Cd84, Icos, Il6st and Il21r) were further tested for direct repression by miR-146a using luciferase reporter assays. The predicted target sequence from Stat1 revealed a 25% repression (Supplementary Fig. 8), comparable to that reported by Rudensky and co-workers24. Similar repression was observed using luciferase reporters containing the minimal target sequences from Icos, Slamf1, Slamf6 or Cd84. All of these targets were specically repressed by miR-146a, but not by a scramble miRNA control (a commercially available, random

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Lymphocytes Ly5a+ cells

Ly5a+ cells B220+ cells CD4+ cells

LPS stimulated

CD11b+

105

52.2

105

104

61.6 3.45

89.5

14.2

104

6.9

103

Ly5a

102

42.7

B220

102

GL-7

102

CD4

102

PD-1

102

12.6

102 103 104 105

0 102 103 104 105

102 103 104 105

Ly5b

CD11b

Fas

CD11b

CXCR5

GC B Non-GC B CXCR5

150

PD-1hi CD11b

2,500

5 0

Relative Il6mRNA

2,000

100

1,500

1,000

500

Mir146a: Mir146a:

Ly5: Ly5:

+/+

a b a b

+/+ +/+ / +/+

a b a b

+/+ +/+ / +/+

a b a b

+/+ +/+ / +/+

a b a b a b a b

+/+ +/+ /

+/+

+/+ /

+/+

All cells Live cells

CD45 CD31 cells

gp38

25 CD35+

gp38+

CD35+

105

+/+ /

104

0.21

0.39

103

Relative Il6mRNA

7AAD

102

CD45

gp38

102

8.4

250K

50K

100K

150K

200K

102 103 104 105

0 102 103 104 105

FSC

CD31

CD21/35

Mir146a :

+/+ /

Unstimulated

IL-6-stimulated Mir146a+/+

IL-6-stimulated Mir146a/

Effector

Nave

3,000

100

2,000

pSTAT3 MFI

1,000

% of max

102 103 104 105

Mir146a :

0 +/+ / +/+ /

pSTAT3

Figure 4 | Contribution of IL-6 signalling to the Tfh and GC B-cell accumulation in miR-146a-decient mice. (ac) Similar expression of Il6 mRNA between miR-146a-decient ( / ) and -sufcient ( / ) cells in SRBC-immunized Ly5a .Mir146a/ : Ly5b .Mir146a / mixed bone marrow

chimeras analyzed 14 weeks post reconstitution. (a) Flow cytometric contour plots showing the gating strategy used to isolate the different cell subsets. (b) Relative amounts of Il6 mRNA in GC B (B220 GL-7 Fas), non-GC B (B220 GL-7 Fas ), Tfh (CD4 CXCR5high PD-1high), and CD11b cells from Ly5a and Ly5b cells analyzed directly after isolation. ND not detectable. The expression of Il6 was normalized to b-actin. (c) Relative amounts of

Il6 mRNA in LPS-stimulated CD11b cells from the same set of chimeric mice as in b. (d,e) No increased expression of IL-6 in miR-146a-decient follicular dendritic cells. (d) Flow cytometric contour plots showing the gating strategy used to isolate follicular dendritic cells. (e) Relative amounts of Il6 mRNA in gp38 and gp38 FDCs (CD45 CD31 CD35 ) from Mir146a / or Mir146a / mice. (f) Histograms and dot plots showing the amounts of

STAT3 phosphorylation in nave and effector CD4 Tcells following acute stimulation with recombinant mouse IL-6. Data are representative of at least two independent experiments. Heights of bar graph in b,c,e represent mean and error bar represents s.d. Each symbol in f represents one mouse and the horizontal bars represent the median values. Statistical signicance was determined using unpaired Students t-test (e) or MannWhitney U-test (f). NS not signicant, *Po0.05, **Po0.01.

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Nave Effector Tfh Nave Effector Tfh Nave Effector Tfh

STAT1 MFI

40 4,000 250

200

150

100

3,000 2,000 1,000

* *

30 20 10

Notch1 MFI

IL21R MFICXCR4 MFI

Mir146a: +/+ / +/+ / +/+ / Mir146a: +/+ / +/+ / +/+ / Mir146a: +/+ / +/+ / +/+ /

Mir146a: +/+ / +/+ / +/+ /

2,000 1,500 1,000

500

1,000

ICOS MFI

SLAMF1 MFI

2,000

1,500

1,000

500

2,500 20,000 15,000 10,000

5,000 0

SLAMF6 MFI

IL6ST MFI

Mir146a: +/+ / +/+ / +/+ /

Mir146a: +/+ / +/+ / +/+

3,000

* * *

NS NS NS

CD84 MFI

2,000 1,500 1,000

500

2,500

2,000

1,500

1,000

500

2,000

Mir146a: +/+ / +/+ / +/+ / Mir146a: +/+ / +/+ / +/+ / Mir146a: +/+ / +/+ / +/+ /

Relative luciferase activity

140

120

100

Relative luciferase activity

140

120

100

140

NS NS

120 **** **** ****

120 ****

miR-146a

Scramble

0 wt-lcos mut-lcos wt-Slamf1 mut-Slamf1 wt-Slamf6 mut-Slamf6

0 wt-II6st mut-II6st

Relative luciferase activity

140

100

Relative luciferase activity

140

120

100

Relative luciferase activity

140

120

100

0 wt-Cd84 mut-Cd84

wt-II21r mut-II21r

Figure 5 | MiR-146a directly represses multiple Tfh-cell mRNAs. (a) Geometric mean uorescence intensity (MFI) of STAT1, CXCR4, Notch1, ICOS, SLAMF1, SLAMF6, CD84, IL-21R and IL6STon nave (CD4 CD44low), effector (CD4 CD44high) and Tfh (CD4 CXCR5high PD-1high Foxp3 ) cells from mice lacking miR-146a ( / ) or wild-type littermates ( / ). Each symbol represents one mouse and the horizontal bars represent the median values.

Data are representative of at least two independent experiments. Statistical signicance was determined using a MannWhitney U-test. (b) Effects of miR-146a (open bars) and scramble (negative control) RNA (lled bars) expression on luciferase reporter constructs containing putative miR-146a target sites as relative luciferase activity (normalized to the Renilla control). Sequence specicity of miRNAmRNA binding was conrmed by introducing mutations to the predicted target sites at regions complementary to the miR-146a seed region. Relative luciferase activity of cells transfected with miR-146a is expressed as a percentage of that transfected with the scramble RNA. Data are representative of at least two independent experiments. The heights of the bars represent the mean, and the error bars represent the s.d. of six technical replicates. Statistical signicance was determined using unpaired Students t-test. NS, not signicant, *Po0.05, **Po0.01, ****Po0.0001.

sequence miRNA mimic; Fig. 5b). Luciferase reporter constructs containing mutations in the sequences recognized by the miR-146a seed region were no longer repressed by miR-146a. This not only validated the sequence specicity of the miRNAmRNA binding but also mapped the miRNA recognition sequence in the 30UTR (Fig. 5b). By contrast, luciferase reporter constructs containing the predicted target sequences from Il21r and Il6st were not repressed by miR-146a (Fig. 5b). Hence, Icos, Slamf1,

Slamf6 and Cd84, but not Il21r and Il6st, are likely to be novel direct targets of miR-146a.

Luciferase assays reveal targets of miRNAs when the miRNA is overexpressed in HEK293T cells. To assess which of the above targets are physiologically relevant and a direct consequence of miR-146a regulation in CD4 T cells (nave, effectors, Treg and Tfh cells), we assessed their protein expression in Ly5a .

Mir146a / : Ly5b .Mir146a / mixed bone marrow

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7436 ARTICLE

chimeras. For this, the geometric mean uorescence intensity was compared between Ly5a and Ly5b cells for each target. Of all targets, the only one that appeared to be controlled cell autonomously by miR-146a in all CD4 subsets examined (including Tfh cells) was ICOS (Fig. 6a). None of the other targets was cell autonomously upregulated in Tfh cells, but some cell-autonomous upregulation was observed in nave CD4 T cells (Supplementary Fig. 9: CXCR4, SLAMF1, SLAMF6 and CD84), effector CD4 T cells (Supplementary Fig. 9: CXCR4) and Tregs (Supplementary Fig. 10: CXCR4, SLAMF1, CD84 and STAT1).

As expected from the luciferase assay results, neither IL6ST nor IL-21R were upregulated in a cell-intrinsic manner in Mir146a / T cells (Supplementary Figs 9 and 10). Collectively, our data suggest that miR-146a acts in T cells to directly repress the expression of Icos, Slamf1, Slamf6, Cd84 and Cxcr4, and of these, the only cell-autonomous effect in Tfh cells is exerted on Icos.

Increased ICOS signalling in T cells causes Tfh-cell accumulation. In light of the profound cell-autonomous control of ICOS by miR-146a and the known roles of ICOS in promoting Tfh-cell differentiation19,48,51, we set out to formally test the contribution of excessive ICOS signalling to the expansion of Tfh cells in the absence of miR-146a. To this end, we rst titrated anti-ICOSL antibody treatment in mice in search of a dose sufciently low so as to partially suppress ICOS signalling without abolishing Tfh-cell formation in wild-type mice. Injections of 25 mg of anti-ICOSL antibody given every 3 days to wild-type mice after immunization with SRBCs had no signicant effect on wild-type Tfh cells (Fig. 6b). We then used this anti-ICOSL treatment regime on 9-week-old Mir146a / and Mir146a / mice immunized with SRBC. This treatment completely and consistently corrected the accumulation of Tfh and GC B cells in Mir146a / mice and still had no effect on wild-type mice (Fig. 6c). Of note, the increase in Tregs seen in Mir146a / mice was not consistently corrected by the anti-ICOSL treatment (Fig. 6c and Supplementary Fig. 11).

We also used a genetic approach to reduce but not eliminate ICOS expression in Mir146a / mice. Icos hemizygosity was achieved by crossing Mir146a / to Icos / mice to generate

Mir146a / Icos / mice. As seen with the antibody blockade, lowering Icos gene dose reduced the percentage of Tfh and GC B cells in Mir146a / Icos / mice to the amount seen in wild-type Mir146a / Icos / mice (Fig. 6d). Importantly, Icos hemizygosity did not rescue Treg-cell accumulation in mice lacking miR-146a (Fig. 6d), suggesting that dysregulation of Treg number is not the cause of Tfh-cell accumulation. Thus, our data indicate that overexpression of ICOS is likely to be a prominent contributor to the Tfh-cell accumulation in Mir146a / mice.

To exclude the possibility that the observed effect of Icos gene dose reduction was a consequence of modulating ICOS expression on Tregs, we co-transferred sorted nave (CD44low CD25 )

CD4 T cells from Mir146a / or Mir146a / mice, which were either Icos/ or Icos / , with Mir146a / Icos /

B cells and Mir146a / Icos / Tregs into Rag1 / recipient mice (Fig. 6e). Mir146a / mice behaved identically to

Mir146a / with no evidence of spontaneous Tfh or GC B-cell accumulation (Supplementary Fig. 12a) or ICOS overexpression (Supplementary Fig. 12b). Fourteen days after adoptive transfer, T cells in all experimental groups showed comparable activation status as measured by the proportion of cells upregulating CD44 or CD25 (Supplementary Fig. 13). Following SRBC immunization, recipients of Mir146a / Icos / T cells had a B50%

reduction in the percentage of Tfh and GC B cells compared with those that received Mir146a / Icos / T cells (Fig. 6f).

Furthermore, halving the gene dose of Icos on Mir146a /

T cells virtually corrected the Tfh-cell accumulation caused by the lack of miR-146a in T cells: there was no statistically signicant difference between the Mir146a / Icos / and Mir146a /

Icos / groups. These results suggest that Icos overexpression in Tfh-cell precursors is indeed responsible, at least in part, for the

Tfh-cell accumulation in miR-146a-decient mice.

MiR-146a limits ICOSL in GC B cells and dendritic cells. The mixed bone marrow chimera results showed that despite the increase in total follicular T cells in the Ly5a .Mir146a / :

Ly5b .Mir146a / chimeras, only the Ly5b .Mir146a / GC B cells, and not the Ly5a .Mir146a / B cells, expanded (Fig. 3c,d). One of the plausible explanations is that miR-146a coordinately regulates a receptorligand pair in Tfh and GC B cells, so overexpression of both T-cell-expressed receptor and GC B-cell ligand (or vice versa) are required for Mir146a / GC B cells to accumulate. Since our data revealed ICOS as the most prominent target of miR-146a in Tfh cells, and T-cell-expressed ICOS interacts with B-cell-expressed ICOS ligand (ICOSL) at different stages of Tfh-cell and GC formation48,51, we assessed whether miR-146a also regulates ICOSL in B cells. Although we did not nd any predicted miR-146a binding site in Icosl 30UTR, previous studies have shown that Icosl transcription is regulated by NF-kB signalling52, which is partly controlled by miR-146a (refs 20,21,23).

To test whether miR-146a controls ICOSL expression in B cells, we sorted total B cells from Mir146a / and Mir146a /

mice, stimulated them in culture for 8 h using lipopolysaccharides (LPS) or anti-CD40 antibody, and compared ICOSL expression on either GC or non-GC B cells. No differences in ICOSL expression were observed in non-GC B cells under any conditions (Fig. 6g). On the other hand, the expression of ICOSL was consistently higher in Mir146a / GC B cellsknown to be dependent on NF-kB signalling5355across all conditions (Fig. 6g). An increase in ICOSL expression was also observed in miR-146a-decient CD11b and CD11c cells following in vitro culture in the absence or presence of LPS (Fig. 6h).

These results suggest that overexpression of ICOSL on GC B cells and myeloid/dendritic cells are likely to enhance the effects of increased ICOS expression on Tfh-cell accumulation. They also suggest that ICOSL overexpression may be required for Mir146a / GC B-cell expansion when ICOS is concomitantly overexpressed in T cells.

Finally, we explored the role of other miR-146a targets that may contribute to the Tfh-cell accumulation. To this end, we investigated the consequences of genetic manipulation of STAT1 that has known Tfh-cell-promoting effects51 and SLAMF1. Mir146a / mice were crossed with mice carrying a loss-of-function mutation in the Stat1 gene (Stat1fae; MGI:3611892) to generate Mir146a / Stat1fae/ mice (Supplementary Fig. 14).

The percentages of Tfh and GC B cells in Mir146a / Stat1fae/ mice were signicantly lower than those in the Mir146a /

Stat1 / mice, but remained about eightfold higher than those seen in wild-type mice (Supplementary Fig. 14). This result suggests that STAT1 contributes to the Tfh-cell accumulation in Mir146a / mice, perhaps by crippling Treg control of Tfh responses or by actions within Tfh cells and/or other cells. We also studied the effect of increased Slamf1 copy number in Tfh-cell formation using NOD.Nkrp1b.Tg(Slamf1)1 (SLAM A6 copies of Slamf1) and NOD.Nkrp1b.Tg (Slamf1)2 (SLAM B44 copies56) mice (Supplementary Fig. 15). No differences were identied in either Tfh-cell or GC formation. Thus, in addition to the regulation of ICOS, miR-146a also represses ICOSL and STAT1 expression, which collectively may contribute to the accumulation of Tfh cells in mice lacking miR-146a.

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DiscussionWe have shown that miR-146a, a microRNA highly expressed in Tfh cells, limits Tfh-cell number. Indeed, in the absence of miR-146a, there was spontaneous expansion of Tfh and GC B

cells. MiR-146a emerges as the rst miRNA to negatively regulate Tfh-cell accumulation. It therefore opposes the action of the miR-17B92 cluster, recently shown to promote Tfh-cell differentiation by repressing Pten, Phlpp2 and Rora mRNAs57,58.

Nave

Effector

Tfh

***

200 150

1,000

NS NS

ICOS MFI

100

4 3 2 1 0

ICOS MFI

800

600

400

200

0 0

ICOS MFI

2,000 1,500

500

1,000

0 +/+ /

a b

+/+ /

a b

+/+ /

a b

Mir146a: Mir146a:

+/+

Mir146a:

+/+

Ly5:

Ly5: Ly5:

2.01.51.00.50.0

2.5

1.0

0.5

0.0

GC B (% B220+ cells)Tregs (% CD4+ cells)

Tfh cells (% CD4+ cells)

GC B cells (% B220+ cells)

2.0

1.5

0 12.5 25 50 100

ICOSL Ab titre (g)

Mir146a:

+/+

+/+ +/+ +/+ /

/ / /

Icos:

ICOS blockade:

GC B cells (% B220+ cells)

** *

20 15 10

5 0

* *

Tregs (% CD4+ cells)

NS NS

Mir146a:

+/+ +/+ / /

Mir146a:

0 +/+ +/+ / /

Mir146a:

+/+

+/+ +/+ / /

0 +/+ +/+ / /

Icos:

+/+

Icos:

+/+

Tfh cells (% CD4+ cells)

anti-CD40

20 NS

Mir146a+/ or

Mir146a/ CD44lo

CD25 CD4+ cells

Mir146a+/+

Icos+/+

Tregs

Mir146a/

B cells

Rag1/

recipients

Icos+/+

Icos+/

14 days

or SRBC

immunization

5 0

Mir146a:

+/ +/ +/ +/

/ / /

Mir146a:

Icos:

+/+ +/+

+/+

/ /

Icos:

GC B cells Non-GC B cells

CD11b+/CD11c+ cells

6,000 ***

***

** **

ICOSL MFI

3,000 4,000

4,000

ICOSL MFI

3,000

2,000

****

2,000

1,000

Mir146a : Mir146a:

Mir146a :

0 +/+ / +/+

+/+ / +/+ +/+

0 +/+

+/+

0 +/+ /

anti-CD40 Isotype

Isotype LPS

LPS Unstimulated

Unstimulated Unstimulated LPS

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Mir146a / T cells might cause the observed heightened sensitivity of these T cells to IL-6 signalling as measured by

STAT3 phosphorylation.

Although this work has focused on identifying Tfh cell-autonomous targets of miR-146a, the milder phenotype observed when only T cells lack miR-146a suggests that T-cell extrinsic factors also contribute to the accumulation of Tfh cells in animals lacking miR-146a. IL-6, which was increased in aged miR-146adecient mice20,23, did not appear to be upregulated in any of the cell types examined in 12- to 16-week-old mice, although a role of IL-6 derived from other haematopoietic cells and/or non-haematopoietic cells cannot be completely excluded.Furthermore, given the signicant effect of IL-6R blockade treatment, it is possible that Mir146a / T cells or myeloid cells do produce increased IL-6 protein in vivo that would not have been detected by our transcriptional studies. On the other hand, antigen-presenting cells that are typically CD11c and/or

CD11b did express increased amounts of ICOSL, which may enhance T-cell priming and exacerbate Tfh-cell accumulation in

Mir146a / mice61. Tfr cells are required to control Tfh cells and GC B cells1517 and have been shown to derive from Foxp3 thymic Tregs, which are less functional in Mir146a / mice due to STAT1 overexpression24. Thus, despite their accumulation, it is likely that Tfr cells are also defective in Mir146a / mice and this may also exacerbate the spontaneous Tfh-cell accumulation.

In addition, increased expression of the costimulatory molecules CD80 (ref. 62) and CD86 (ref. 63) and the reported enhanced sensitivity of T cells to antigen stimulation21 due to dysregulated NF-kB activation may also contribute to the Tfh-cell expansion.

It has been reported previously that strong TCR signalling can skew the differentiation pathway of nave CD4 T cells towards the Tfh lineage64,65. Collectively, all of these miR-146a-mediated effects in various cell types are likely to act cooperatively to cause the accumulation of Tfh cells.

In conclusion, this report identies miR-146a as an important negative regulator of Tfh and GC B-cell accumulation. It is likely that this contributes to maintaining immunological tolerance and preventing the autoimmune phenotypes observed in mice lacking this microRNA. These ndings on miR-146a-mediated control of Tfh and GC B-cell number may therefore improve our understanding of human autoimmunity and inammatory syndromes.

Note added in proof: A role for miR-146a in repressing miR-155-induced Tfh-cell accumulation was reported while this work was under consideration66.

Methods

Mice and immunizations. The mice used were C57BL/6 (B6), B6.Ly5a, B6.Rag1 / , B6.Cd28 / , B6.OT-II, B6.Mir146a / (ref. 20), B6.Stat1fae/,

Figure 6 | Increased ICOS and ICOSL expression is likely to be the major driver of Tfh-cell accumulation in the miR-146a-decient mice.(a) Geometric mean uorescence intensity (MFI) of ICOS on nave, effector CD4 and Tfh cells from Mir146a /: Mir146a / and Mir146a /:

Mir146a/ mixed bone marrow chimeras 16 weeks post reconstitution. (b) Percentages of Tfh cells in wild-type mice immunized with sheep red blood cells (SRBC) and treated with different amounts of anti-ICOSL blocking antibody on days 0, 3 and 6 post immunization. Each symbol represents the mean and error bars represent the s.d. of three biological replicates. (c) Percentages of Tfh, GC B and Treg cells in 9-week-old wild-type ( / ) and

miR-146a-decient mice ( / ) treated with 25 mg anti-ICOSL blocking antibody or PBS control on day 0, 3 and 6 following SRBC immunization.

(d) Percentages of Tfh, GC B and Treg cells in 9-week-old Mir146a / Icos /, Mir146a / Icos / , Mir146a / Icos /, Mir146a / Icos/ mice following SRBC immunization. (e,f) Adoptive transfer of puried nave (CD44low CD25 ) CD4 T cells from either Mir146a/ Icos/ ,

Mir146a/ Icos/ , Mir146a / Icos/ or Mir146a / Icos/ , together with Mir146a / Icos / B cells and Mir146a/ Icos/ Tregs into Rag1 / recipients according to the strategy shown in e. The recipient mice were immunized with SRBC 14 days after transfer and killed 7 days post immunization. (f) Percentages of Tfh and GC B cells in Rag1 / recipient mice following adoptive transfer and SRBC immunization. (g,h) ICOSL MFI on GC

B (g, left), non-GC B (g, right) and a mixture of CD11b and CD11c cells (h) from SRBC-immunized wild-type ( / ) or miR-146a-decient ( / )

mice following stimulation with LPS, anti-CD40 antibody or isotype control. Heights of bars represent the mean and error bars represent s.d. of three technical replicates. Each symbol represents one mouse and the horizontal bars represent the median values. Data are representative of at least two independent experiments. Statistical signicance was determined using paired Students t-test (a), unpaired Students t-test (b,g,h) or MannWhitney U-test (c,d,f). NS, not signicant, *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001.

Our luciferase assays, together with published reports, have shown that ectopic expression of miR-146a can lead to repression of multiple Tfh-cell-expressed mRNAs, including Icos, Slamf1, Slamf6, Cd84, Stat1, Cxcr4 and Notch1. Although all of the above targets were upregulated in T cells from miR-146a-decient mice, ICOS was the only target upregulated cell autonomously in Tfh cells and the most robustly upregulated across all T-cell subsets examined. Strikingly, dampening ICOS-mediated signalling in Mir146a / mice, either by generating Mir146a / Icos /

mice or by injecting a suboptimal dose of anti-ICOSL antibody, completely prevented miR-146a-driven Tfh-cell accumulation. Furthermore, adoptive transfer experiments in which only non-Tregs lacked one allele of Icos demonstrated that repressing ICOS signalling in Tfh-cell precursors themselves, rather than in Tregs, is required to control Tfh-cell number. This is consistent with previous reports showing that cell-autonomous Icos mRNA overexpression due to defective Roquin function contributes to excessive Tfh-cell number19.

We have also found that miR-146a acts cell autonomously in GC B cells to limit their number. Increased expression of ICOSL on GC B cells is a plausible contributor to their accumulation in the absence of miR-146a, and may explain why despite the striking increase of Mir146a / Tfh cells in Mir146a/: Mir146a /

chimeras only Mir146a / GC B cells expanded. Limiting ICOSL signalling is likely to be important to prevent excessive B-cell activation. Indeed, mice overexpressing soluble ICOSL-Fc developed hypergammaglobulinemia and plasmacytosis59. Our results suggest that concomitant control of ICOS on Tfh cells and ICOSL on GC B may be necessary to control the number of GC B cells.

In addition to ICOSICOSL regulation, miR-146a-mediated repression of other T-cell-expressed targets might also contribute to limit Tfh cells. Our data suggest STAT1 is one such target: lowering the dose of STAT1 had a signicant corrective effect on Tfh-cell accumulation. This effect is likely to, at least in part, be a consequence of STAT1 overexpression in Treg cells, including Tfr cells15, leading to impaired repressive ability24. In addition, STAT1 is known to function downstream of several Tfh-inducing cytokines, including IL-6, IL-12, IL-21 and IL-27 (refs 3,51,60), thus STAT1 overexpression may enhance the sensitivity of miR-146a-decient T cells to stimulation by these cytokines and increase their propensity to differentiate along the Tfh lineage. Nevertheless, we did not observe an increase in IL-6-induced STAT1 phosphorylation in Mir146a / T cells. Another candidate that may contribute to the Tfh-cell accumulation in miR-146a-decient mice is IL6ST. We found a subtle increase in IL6ST expression on Mir146a / T cells, although our luciferase assay data indicate that it is unlikely to be a direct target of miR-146a. We speculate that increased IL6ST expression in

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B6.Icos / (Australian Phenomics Facility, ANU), NOD.Nkrp1b.Tg(Slamf1)1 (ref. 56) and NOD.Nkrp1b.Tg(Slamf1)2 (ref. 56; Immunogenetics Research Facility,

James Cook University). Male and female mice were used in experiments at approximately equal ratio between the wild-type/untreated and mutant/treated groups. Mir146a / mice were provided by David Baltimore (Caltech, USA).

Stat1-face (Stat1fae) mice were derived from an N-ethyl N-nitrosourea mutagenesis screen67 and kindly donated by Christopher C. Goodnow (ANU, Australia). Mir146a / , Mir146a / Stat1fae/ , NOD.Nkrp1b.Tg(Slamf1)1 (ref. 56) and

NOD.Nkrp1b.Tg (Slamf1)2 (ref. 56) mice were taken down at 12 to 16 weeks old unless otherwise stated. All mice were housed under a specic pathogen-free environment. All experiments were approved by the Australian National University Animal Experimentation Ethics Committee and the James Cook University Animal Ethics Committee. To generate thymus-dependent GC responses, where mentioned, mice were immunized i.v. with 2 108 SRBCs (Applied Biological

Products Management, Australia) and were taken down at day 7 post immunization.

Human tonsils. Human tonsils were obtained from consenting patients undergoing routine tonsillectomy at the Canberra Hospital (Canberra, Australia) with the approval from the institutional human research ethics committee. Tonsillar cells were isolated by mechanical disruption and Ficoll-Paque density gradient centrifugation.

Adoptive transfers. To study the kinetics of miR-146a expression in a thymus-dependent GC response, 5 105 sorted nave OT-II CD4 T cells were adoptively

transferred into Ly5a wild-type B6 recipient mice via i.v. injection. To limit miR-146a deciency to T cells, 1 105 sorted nave Mir146a / OT-II or

Mir146a / OT-II T cells were adoptively transferred into Cd28 / recipient mice. The recipients were immediately immunized i.p. with 100 mg OVA (Sigma) precipitated in alum. To study the contributions of miR-146a deciency in T and B cells, 4 106 CD4 T cells and 6 106 B cells from Mir146a / or

Mir146a / mice were adoptively transferred into Rag1 / mice. The recipients were immunized with SRBC 14 days after transfer and they were killed 7 days post immunization. In addition, to examine the contribution of ICOS overexpression specically in Tfh-cell precursors to the Tfh-cell accumulation, 7.5 105 CD44low

CD25 CD4 T cells from either Mir146a / Icos / , Mir146a / Icos / , Mir146a / Icos / or Mir146a / Icos / donor mice were co-transferred with 3 106 Mir146a / Icos / B cells and 7.5 104 Mir146a / Icos /

Tregs into Rag1 / recipients. The recipients were bled (to assess the activation status of T cells) at day 14 following adoptive transfer and immediately immunized with SRBC. The mice were then killed 7 days post immunization.

Antibodies. Antibodies and streptavidin conjugates used for ow cytometry were (respective vendors, catalogue numbers and dilutions at which they were used are shown in parentheses): anti-mouse Ly5a-Pacic Blue (Biolegend, 110722, 1:200), Ly5a-Alexa Fluor 700 (BD, 561235, 1:200), Ly5b-Alexa Fluor 700 (BD, 560693, 1:200), Ly5b-PerCP Cy5.5 (BD, 552950, 1:200), CD4-Alexa Fluor 700 (BD, 557956, 1:800), CD4-APC Cy7 (BD, 552051, 1:800), B220-PerCP (BD, 553093, 1:800), CXCR5-biotin (BD, 551960, 1:50), PD-1-PE (BD, 551892, 1:100), PD-1-Brilliant Violet 421 (BD, 562584, 1:400), CD25-Alexa Fluor 647 (BD, 563598, 1:100), CD44-APC (BD, 559250, 1:400), CD44-Alexa Fluor 700 (BD, 560567, 1:400), CD44-PE CF594 (BD, 562464, 1:400), Foxp3-FITC (eBioscience, 11-5773-82, 1:400), Foxp3-eFluor 450 (eBioscience, 48-5773-82, 1:400), GL-7-FITC (BD, 553666, 1:400), Fas-PE Cy7 (BD, 557653, 1:400), Vb5.1/5.2-FITC (BD, 562087, 1:400), ICOSL-PE (Biolegend, 107405, 1:400), CD11b-APC Cy7 (BD, 557657, 1:200), CD11c-PerCP Cy5.5 (BD, 560584, 1:200), IFN-g-FITC (BD, 554411, 1:400),

IL-4-PE Cy7 (BD, 560699, 1:100), IL-17A-Alexa Fluor 700 (BD, 560820, 1:100), STAT1 (pY701)-Pacic Blue (BD, 560310, 1:5), STAT3 (pY705)-PE (BD, 562072, 1:20), STAT1-Alexa Fluor 647 (BD, 558560, 1:40), CXCR4-biotin (BD, 551968, 1:200), Notch1-PE (BD, 552768, 1:200), ICOS-PE (BD, 552146, 1:200), SLAMF1-PE (BD, 562651, 1:200), SLAMF6-Alexa Fluor 647 (BD, 561547, 1:100), CD84-PE (Biolegend, 122806, 1:200), IL-21R-APC (Biolegend, 131910, 1:100), gp130/IL6STAPC (R&D Systems, FAB4681A-100, 1:100), CD31-PE (BD, 561073, 1:200), gp38-Alexa Fluor 488 (eBioscience, 53-5381-82, 1:200), CD21/35-biotin (BD, 562796, 1:200), anti-human CD4-APC Cy7 (BD, 557871, 1:800), CXCR5-Alexa Fluor 647 (BD, 558113, 1:100), PD-1-PE (eBioscience, 12-9969-42, 1:200), CD25-PE Cy7 (Biolegend, 356108, 1:100), CD44-Alexa Fluor 700 (BD, 560567, 1:400), and streptavidin-PE Cy7 (BD, 557598, 1:800). Monoclonal blocking antibody against ICOSL (clone MIL-5733)68 was provided by A.H. (DRFZ, Germany) and blocking antibody against IL-6R (catalogue number BE0047, 150 mg per mouse) was purchased from BioXCell. Mouse anti-CD40 (catalogue number BE0016-2, 10 mg ml 1) antibody was also purchased from BioXCell.

Bone marrow chimeras. Chimeras were generated by sub-lethally irradiating Rag1 / recipient mice (500 cGy, X-rad) and reconstituting their immune system by transferring 4 106 bone marrow cells from donor mice. Chimeric mice were

taken down 16 weeks post reconstitution unless otherwise stated.

Cell isolation and stimulation. Single-cell suspensions were prepared from spleens of unimmunized and/or immunized mice. Suspensions were prepared in RPMI 1640 media (Gibco) supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 U penicillinstreptomycin (Gibco), 2 mM L-Glutamine (Gibco), 10 mM HEPES (Sigma), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Sigma), 55 mM 2-mercaptoethanol (Gibco) by mechanical disruption and gentle pipetting through 70 mm nylon mesh lters (BD Biosciences). For cytokine staining (IFN-g, IL-4 and IL-17), cells were cultured for 4 h in a 37 C/5% CO2 incubator with 50 ng ml 1 PMA (Sigma) and 1 mM ionomycin (Sigma) in the presence of Golgi Stop (BD Biosciences). For ICOSL induction, B220 cells and combined CD11b /CD11c cells were isolated from SRBC-immunized

Mir146a / or Mir146a / mice by negative and positive selection, respectively, using MACS columns (Miltenyi Biotec) according to the manufacturers instructions. About 5 105 B cells or CD11b /CD11c cells were cultured per well in a

96-well plate (Corning) with different conditions: complete RPMI only (unstimulated), 1 mg ml 1 LPS from Escherichia coli (Sigma), 10 mg ml 1 rat IgG2a isotype control antibody (BioXCell) or 10 mg ml 1 rat anti-mouse CD40 antibody (BioXCell) for 8 h in a 37 C/5% CO2 incubator. To induce IL-6 expression, puried CD11b cells from mixed bone marrow chimeras were stimulated overnight with 100 ng ml 1 LPS (Sigma) in a 37 C/5% CO2 incubator. To study the responsiveness of T cells to IL-6 signalling, total splenocytes were stimulated with recombinant mouse IL-6 (20 ng ml 1) for 15 min in a 37 C/5% CO2 incubator and levels of STAT3 phosphorylation were analyzed according to published protocols69.

Flow cytometry. For lymphocyte analysis, single-cell suspensions were prepared as described in the previous section. For myeloid cell-population analysis, spleens were treated with Collagenase P (Roche) and DNase I (Sigma) before preparing single-cell suspensions for staining. FDCs were isolated as described previously35. Briey, spleens were digested in 1 ml dissociation media (complete RPMI, 1 mg ml 1 collagenase D, 200 mg ml 1 dispase and 100 mg ml 1 DNase I) for30 min in a 37 C, 5% CO2 incubator. Cell suspensions were then gently triturated through P1000 pipette tips and incubated for a further 30 min. Dissociated cells were washed with complete RPMI to remove excess digestive enzymes. To stain for surface markers, cells were incubated with each antibody layer for 30 min in the dark at 4 C and washed thoroughly with ice-cold FACS buffer (2% FCS and 0.1% NaN3 in PBS) between each layer. Intracellular staining was performed using the

Intracellular Staining Kit (eBioscience) according to the manufacturers instructions. Serum IL-6 was measured using CBA bead assays (BD Biosciences) following the manufacturers instructions. A BD LSRII Flow Cytometer with FACSDiva software were used for ow cytometry acquisition and FlowJo (Tree Star) was used for analysis. FACS Aria (BD Biosciences) was used for cell sorting, cells were collected in FCS. Tonsillar T cells were sorted using the following strategy: CD4 CD25 CD44low CXCR5int/low PD-1int/low nave T cells, CD4

CD25 CD44high CXCR5int/low PD-1int/low non-Tfh effector cells, CD4 CD25 CD44int CXCR5int/low PD-1int/low Tregs, CD4 CD25 CXCR5high PD-1high Tfh cells, CD4 CD25 CXCR5high PD-1high Tfr cells.

Luciferase reporter assay. HEK293T cells cultured in DMEM media (Gibco) supplemented with 10% heat-inactivated FCS, 100 U penicillinstreptomycin (Gibco) and 2 mM L-Glutamine (Gibco) in 24-well plates were co-transfected with pmirGLO (Promega) luciferase reporter plasmids containing either wild-type or mutated putative miR-146a minimal target sequences (Supplementary Table 1; designed according to pmirGLOs protocol) and mirVana miR-146a mimic(Life Technologies) or mirVana negative control #1 (scramble; Life Technologies) using Lipofectamine 2000 (Life Technologies) according to the manufacturers instructions. The luciferase construct containing Stat1 30UTR was purchased from Genecopoeia. After 24 h, 3 105 transfected cells were moved into opaque

at-bottom 96-well plates and incubated for another 24 h. Cells were then harvested and luciferase activity was assessed using Dual-Glo Luciferase Assay System (Promega) according to manufacturers protocol.

Microarray. Total RNA extraction from sorted tonsillar cells was done using the RNeasy Kit (QIAGEN) according to the manufacturers protocol. RNA quantity and quality was determined using a Nanodrop 1000 and an Agilent 2100 Bioanalyzer, respectively. Hybridization on Agilent Human miRNA Microarray (V1) Kit was performed at The Ramaciotti Center for Genomics (University of New South Wales, Australia). Triplicates (for human miRNA array) were collected and averaged in GeneSpring. The microarray data has been deposited in NCBIs Gene Expression Omnibus and is accessible through GEO Series accession number GSE64833.

Real-time PCR analysis. RNA was extracted using Trizol reagent (Life Technologies) according to the manufacturers protocol, and complementary DNA was synthesized using the miScript II reverse transcription Kit (QIAGEN) following the manufacturers instructions. Quantitative PCR to detect for miR-146a expression was performed using the miScript SYBR Green PCR Kit (QIAGEN) and miScript Primer Assays (QIAGEN) to amplify mature Mir146a and the U6 small nuclear RNA. Il6 and b-actin mRNAs were amplied using specic primer pairs

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purchased from Origene Technologies(Il6F: 30-TACCACTTCACAAGTCGGA GGC-50; Il6R: 30-CTGCAAGTGCATCATCGTTGTTC-50; ActinF: 30-CATTGC TGACAGGATGCAGAAGG-50; ActinR:30-TGCTGGAAGGTGGACAGTG AGG-50). Quantitative PCR was performed on an Applied Biosystems 7900 machine. The expression of Mir146a was normalized to U6, while the expression of Il6 was normalized to b-actin. The gene specic expression change was calculated using the 2 DDCt method.

Statistical analysis. Statistical signicance was determined using the non-parametric MannWhitney U-test unless specied otherwise. The sample size was chosen so that it would be possible to conduct MannWhitney test with at least 95% condence. Consequently in most experiments, at least ve mice per group were used. All statistical analyses were performed using the GraphPad Prism software. NS, not signicant, *Po0.05, **Po0.01, ***Po0.001, ****Po0.0001.

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Acknowledgements

We thank M. Devoy and H. Vohra (ANU) for technical assistance on uorescence-

activated cell sorting, R.A. Sweet, R.J. Rigby, P. Gonzalez and I.A. Parish (ANU) for

technical assistance in several experiments, H. Qi and D. Liu (Tsinghua University,

China) for helpful discussions and for providing the stimulation protocols to induce

ICOSL expression, and C. C. Goodnow and A. Yates (ANU) for kind provision and

initial characterization of the Stat1fae mice. This work was funded by the National Health

and Medical Research Council (NHMRC) program and project grants and Elizabeth

Blackburn Fellowship to C.G.V., International Postgraduate Research Scholarship

to A.P., NHMRC/MSRA Betty Cuthbert Fellowship to M.A.J., National Research

Service Award F30HL110691 and UCLA/Caltech Medical Scientist Training Program

to J.L.Z.

Author contributions

A.P. designed and performed experiments, analyzed the data and wrote the manuscript.

M.S. performed the human miRNA array and miR-146a-decient mouse microarray

experiments and analyzed the data. N.J.W., I.P., S.K.L. performed experiments. X.T.D.

and M.A.J. performed experiments on Slamf1 transgenic mice, analyzed the data and

reviewed the manuscript. A.H. contributed the anti-ICOSL antibody and reviewed the

manuscript. J.L.Z. reviewed the manuscript. R.C. contributed data on the transcription

factor analysis and reviewed the manuscript. V.A. performed experiments and reviewed the

manuscript. C.G.V. designed experiments, wrote the manuscript and supervised the study.

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signalling to limit accumulation of T follicular helper cells and germinal centers.

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Tight control of T follicular helper (Tfh) cells is required for optimal maturation of the germinal centre (GC) response. The molecular mechanisms controlling Tfh-cell differentiation remain incompletely understood. Here we show that microRNA-146a (miR-146a) is highly expressed in Tfh cells and peak miR-146a expression marks the decline of the Tfh response after immunization. Loss of miR-146a causes cell-intrinsic accumulation of Tfh and GC B cells. MiR-146a represses several Tfh-cell-expressed messenger RNAs, and of these, ICOS is the most strongly cell autonomously upregulated target in miR-146a-deficient T cells. In addition, miR-146a deficiency leads to increased ICOSL expression on GC B cells and antigen-presenting cells. Partial blockade of ICOS signalling, either by injections of low dose of ICOSL blocking antibody or by halving the gene dose of Icos in miR-146a-deficient T cells, prevents the Tfh and GC B-cell accumulation. Collectively, miR-146a emerges as a post-transcriptional brake to limit Tfh cells and GC responses.

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Title

MicroRNA-146a regulates ICOS-ICOSL signalling to limit accumulation of T follicular helper cells and germinal centres

Author

Pratama, Alvin; Srivastava, Monika; Williams, Naomi J; Papa, Ilenia; Lee, Sau K; Dinh, Xuyen T; Hutloff, Andreas; Jordan, Margaret A; Zhao, Jimmy L; Casellas, Rafael; Athanasopoulos, Vicki; Vinuesa, Carola G

Pages

6436

Publication year

2015

Publication date

Mar 2015

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms7436

ProQuest document ID

1660931402

MicroRNA-146a regulates ICOS-ICOSL signalling to limit accumulation of T follicular helper cells and germinal centres

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