Cutting Edge: The Orphan Nuclear Receptor Nr4a1 Regulates CD8⁺ T Cell Expansion and Effector Function through Direct Repression of Irf4

Abstract

The transcription factor IFN regulatory factor (IRF)4 was shown to play a crucial role in the protective CD8⁺ T cell response; however, regulation of IRF4 expression in CD8⁺ T cells remains unclear. In this article, we report a critical role for Nr4a1 in regulating the expansion, differentiation, and function of CD8⁺ T cells through direct transcriptional repression of Irf4. Without Nr4a1, the regulation of IRF4 is lost, driving an increase in Irf4 expression and, in turn, resulting in a faster rate of CD8 T cell proliferation and expansion. Nr4a1-deficient mice show increases in CD8 T cell effector responses with improved clearance of Listeria monocytogenes. Our data support a novel and critical role for Nr4a1 in the regulation of CD8⁺ T cell expansion and effector function through transcriptional repression of Irf4.

Introduction

The NR4A subfamily of orphan nuclear receptors within the steroid thyroid receptor family includes Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (NOR-1). These nuclear receptors function as transcription factors to either induce or repress gene transcription (1, 2). In the thymus, Nr4a1 is activated as part of the immediate-early response downstream of TCR signaling (3, 4) and serves as an indicator of the strength of TCR signals (5, 6).

The effector differentiation of CD8⁺ T cells is regulated by various transcription factors (7). IFN regulatory factor (IRF)4, a member of the IRF family of transcription factors, was recently shown to be vital for sustaining the expansion and effector differentiation of CD8⁺ T cells (8–10). IRF4-deficient CD8 T cells proliferated less, were more prone to apoptosis, and produced fewer effector molecules, such as IFN-γ (11). IRF4-deficient mice cannot clear the intracellular bacterial pathogen Listeria monocytogenes because of intrinsic defects in CD8 T cell expansion and effector function (12). Furthermore, it was found that IRF4-transduced CD8 T cells exhibited increased proliferation, suggesting that ectopic expression of IRF4 promotes polyclonal CD8 T cell expansion (13).

In the current study, we found that the absence of Nr4a1 in mice resulted in an increase in the expression of Irf4, as well as the frequency of IRF4⁺ CD8⁺ T cells, in both the thymus and periphery. The rise in IRF4 resulted in increased proliferative behavior and effector differentiation of CD8⁺ T cells. Using chromatin immunoprecipitation (ChIP) and a luciferase promoter-reporter assay, we found that, mechanistically, Nr4a1 functions to inhibit the expression of Irf4 by directly binding to the Irf4 promoter region. To further demonstrate this regulation, we show that small interfering RNA (siRNA)-mediated knockdown of Nr4a1 in CD8 cells in vitro increased the expression of Irf4, whereas overexpression of Nr4a1 in Nr4a1-deficient CD8⁺ T cells decreased the expression of Irf4. Furthermore, we found that dysregulation of Irf4 in Nr4a1-deficient CD8⁺ T cells increased the development of committed effector cells, IFN-γ production, and L. monocytogenes clearance upon infection. Therefore, Nr4a1 plays a critical role in regulating the proliferative potential and function of effector CD8 T cells through the transcriptional repression of IRF4.

Materials and Methods

Animals

C57BL/6J wild-type (WT) mice (000664), C57BL/6-Tg(TcraTcrb)1100Mjb/J (003831) OT1 mice, B6.SJL-Ptprc^a Pepc^b/BoyJ (002014) CD45.1 mice, and Nr4a1^−/− mice on a congenic C57BL/6J background (006187) were from The Jackson Laboratory. Mice were fed a standard rodent chow diet and were housed in microisolator cages in a pathogen-free facility.

Flow cytometry and Abs

Thymi and lymph nodes were excised and pushed through a 70-μm strainer. Cells (2 × 10⁶–4 × 10⁶) were resuspended in staining buffer (1% BSA [w/v] and 0.1% [w/v] sodium azide in PBS). LIVE/DEAD Fixable Dead Cell Stain (Invitrogen) was used for analysis of viability. For intracellular cytokine staining, cells were stimulated for 2 h with PMA (50 ng/ml) and ionomycin (1 g/ml; Sigma-Aldrich) in the presence of brefeldin A (GolgiPlug; BD Biosciences). For additional intracellular staining, cells were fixed and made permeable with the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences). CXCR3-allophycocyanin, CD69-PerCP, IRF4-allophycocyanin, CD44–Alexa Fluor 700, CD4-allophycocyanin, CD4-FITC, CD8-PE Texas Red, CD8 PerCP-Cy5.5, and TCRβ–allophycocyanin–eFluor 780 were purchased from eBioscience or BD Pharmingen.

Adoptive transfers

CD8⁺ T cells were isolated with an EasySep Mouse CD8 Enrichment Kit, according to the manufacturer’s instructions (STEMCELL Technologies). CD8 cells were labeled with 2.5 μM CFSE (Molecular Probes). A total of 1 × 10⁷ cells in 200 μl PBS was injected i.v. into unirradiated recipient mice.

Quantitative real-time PCR

Thymocyte and lymphocyte cell populations were isolated by flow cytometry, and total cellular RNA was collected with an RNeasy Plus Micro Kit, according to the manufacturer’s protocol (QIAGEN). cDNA was used for each real-time condition with a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) and TaqMan Gene Expression MasterMix and TaqMan primers. Data were analyzed and presented based on the relative expression method.

Transfection of plasmids

Cells were treated with plasmid or siRNA complexes using Lipofectamine 2000 in Opti-MEM media (both from Invitrogen), following the manufacturer’s instructions. The specific silencing of target genes was confirmed by quantitative real-time PCR.

ChIP assay

A total of 2 × 10⁷ CD8⁺ T cells, isolated from peripheral lymph nodes using a CD8⁺ selection kit (EasySep), was activated with plate-bound anti-CD3,CD28 overnight. CD8⁺ cells were transfected with DDK(flag)-tagged Nr4a1 and cross-linked with 2 mM DSG (Sigma) and 1% formaldehyde, methanol-free (Thermo Scientific Pierce). Chromatin was sheared by sonication in a Bioruptor sonicator (Diagenode). Immunoprecipitation was carried out, and DNA was isolated. Target genes were amplified from the isolated DNA using SYBR Green Master (Roche).

L. monocytogenes infection

Mice were infected i.p. with 3000 CFU the L. monocytogenes strain recombinant for OVA. Bacterial titers were quantified as previously described (14).

Statistical analysis

Data were analyzed with the Student t test, or with one-way ANOVA for comparison of more than one group, using Prism4 (GraphPad Software).

Results and Discussion

Nr4a1-deficient mice exhibit increased IRF4 expression and CD8 T cell proliferation

We first analyzed the expression of Irf4 in sorted TCRβ⁺CD8⁺CD4⁻ T cells from thymus and lymph nodes of C57BL6/J (B6) and Nr4a1^−/− mice by quantitative PCR (Fig. 1A, Supplemental Fig. 1A). In the absence of Nr4a1, we found a significant increase in mRNA expression of Irf4 in TCRβ⁺CD8⁺CD4⁻ T cells. We analyzed protein expression of IRF4 in mature TCRβ⁺CD8⁺CD4⁻ T cells found in the thymus and lymph nodes. We found an elevation in IRF4, as well as an increase in the frequency of TCRβ⁺CD8⁺IRF4⁺ T cells, in Nr4a1-deficient mice (Fig. 1B, Supplemental Fig. 1C). Recent studies showed that IRF4-deficient CD8⁺ T cells fail to expand and accumulate compared with control CD8⁺ T cells (11). In the case of high and prolonged expression of IRF4, CD8⁺ T cells exhibited greater expansion after stimulation (13). We reasoned that, in the case of Nr4a1-deficient CD8⁺ T cells, where IRF4 is upregulated, CD8⁺ T cells would proliferate in a more robust manner than their Nr4a1-intact counterparts. We first stimulated CFSE-labeled CD8⁺ T cells with different concentrations of anti-CD3 for 60 h in vitro (Fig. 1C). In the absence of Nr4a1, CD8⁺ T cells proliferated at a faster rate upon CD3 stimulation. We stimulated CFSE-labeled CD8⁺ T cells isolated from either OT1 or Nr4a1^−/−OT1 peripheral lymph nodes with the OVA peptide SIINFEKL (Fig. 1D). Ag-specific stimulation also resulted in an increased proliferative response. To determine whether Nr4a1 functions to regulate proliferation in peripheral CD8⁺ T cells upon Ag stimulation, we knocked down Nr4a1 using siRNA in OT1 CD8 T cells (Fig. 1E). The ablation of Nr4a1 gene expression resulted in increased proliferation after stimulation. Previous studies determined that ectopic expression of IRF4 strongly promoted the expansion of OT1 CD8 T cells (8, 10, 11, 13). We clearly demonstrate that the Nr4a1-deficient CD8 T cell population, which has increased expression of IRF4, exhibits faster rates of proliferation upon stimulation.

FIGURE 1.

Nr4a1-deficient mice exhibit increased CD8 T cell proliferation and IRF4 expression. Quantitative PCR measurement of the levels of Irf4 RNA relative to Gapdh from sorted thymic and lymph node populations (A) and expression of Irf4 on TCRβ⁺CD8⁺ lymphocytes from the thymus (left panel) and lymph nodes (right panel) (B) of Nr4a1^−/− and WT age- and sex-matched controls. At 60 h after in vitro anti-CD3 stimulation, TCRβ⁺CD8⁺ lymphocytes were analyzed for CFSE expression levels (C) and CFSE expression of OT1.CD8⁺ and OT1.Nr4a1^−/− CD8⁺ T cells isolated from the lymph nodes and plated in vivo with OVA for 2 d (D). (E) CFSE expression of OT1.CD8⁺ and OT1CD8⁺ T cells + Nr4a1 siRNA isolated from the lymph nodes and plated in vitro with OVA for 2 d. Data are representative of two separate experiments with at least three age- and sex-matched mice/group; horizontal lines denote the mean. *p < 0.05, unpaired two-tailed t test.

Nr4a1 directly controls induction of Irf4

To determine mechanistically how NR4A1 regulates IRF4, we reduced Nr4a1 expression using siRNA in B6 CD8⁺ T cells isolated from peripheral lymph nodes. We activated the CD8 T cells with anti-CD3,CD28 over the course of siRNA administration. After 72 h, we found a nearly 70% reduction in Nr4a1 expression (Supplemental Fig. 1D) and a corresponding 30–40% increase in Irf4 in the siRNA-treated cells (Fig. 2A). We next overexpressed Nr4a1 in B6 and Nr4a1^−/− CD8⁺ T cells isolated from peripheral lymph nodes by transfecting the cells with Flag-tagged Nr4a1 (Supplemental Fig. 1E). Analysis of Irf4 by quantitative PCR revealed a decrease in the expression on Irf4 upon transfection of Nr4a1 in Nr4a1-deficient CD8 T cells (Fig. 2B).

FIGURE 2.

Nr4a1 directly controls induction of Irf4. (A) Quantitative PCR measurement of the levels of Irf4 RNA relative to Gapdh in αCD3,CD28–activated CD8 single-positive lymphocytes from B6.WT mice, WT mice treated by siRNA Nr4a1 knockdown, and Nr4a1^−/− mice. (B) Transfection of Nr4a1.Flag tagged into WT and Nr4a1^−/− anti-CD3,CD28 activated CD8 single-positive lymphocytes and quantitative PCR analysis of Irf4 expression upon transfection. (C) ChIP of Nr4a1 at the Irf4 promoter in activated CD8 single-positive lymphocytes assessed in untransfected (control) and transfected CD8. (D) A luciferase promoter assay was performed with RAW cells. The cells were transfected with the 2-kb promoter region of Irf4 and an empty vector or the Nr4a1 open reading frame. Nearly 18 h posttransfection, luciferase activity was measured. Data are representative of two separate experiments with at least three mice/group. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005, unpaired two-tailed t test.

The IRF4 promoter contains putative binding sites for Nr4a1 (at −925, −1075, −1555, −2375, and −2565 from the start site of transcription). Therefore, we performed ChIP and found that Nr4a1 binds directly to the promoter region of Irf4, clearly demonstrating that Nr4a1 directly controls the expression of Irf4 (Fig. 2C). We next performed an Irf4 luciferase promoter-reporter assay (Fig. 2D). We transfected RAW cells with the 3.8-kb promoter region of Irf4, together with an empty control vector or with increasing amounts of a vector containing the Nr4a1 open reading frame. Our data clearly demonstrate that Nr4a1 functions to directly repress Irf4 expression. Furthermore, we verified that Nr4a2 and Nr4a3 do not play a redundant role in CD8 T cells, because we found little to no expression of these transcription factors (Supplemental Fig. 1B).

We (15) and other investigators (16, 17) demonstrated that Nr4a1 functions to inhibit gene expression by direct binding to the gene promoter and recruitment of the CoREST corepressor complex. Therefore, Nr4a1 likely functions to directly repress Irf4 expression through binding of the Irf4 promoter region, followed by recruitment of the CoREST corepressor complex.

Nr4a1^−/− CD8⁺ T cells display altered proliferative behavior

We next examined whether Nr4a1 plays a role in the optimal proliferation and survival of CD8⁺ T cells in vivo. To do so, we first administered BrdU (1 mg/mouse) in vivo over the course of 12 h and analyzed BrdU incorporation after 24 h (Fig. 3A, Supplemental Fig. 2A, 2B). Consistent with our in vitro observations, a higher percentage of CD8⁺ T cells isolated from the lymph nodes of Nr4a1^−/− mice incorporated BrdU, indicating that the absence of Nr4a1 results in an aberrant increase in the level of homeostatic proliferation. Nr4a1^−/− CD8⁺ T cells were found to express elevated levels of Irf4 directly ex vivo. It was reported previously that IRF4 sustains proliferation by repressing the production of CDK inhibitors and Bim (8, 10, 11, 13), suggesting that elevated levels of IRF4 in Nr4a1-deficient CD8⁺ T cells function to repress these factors, which results in the increase in homeostatic proliferation. We showed previously that there was no difference in apoptosis between Nr4a1^−/− and B6 mice (15) and confirmed this finding (Supplemental Fig. 2G). We analyzed the expression of two CDK inhibitors, Cdkn1a and Cdkn1c, in CD8⁺ T cells isolated from peripheral lymph nodes of B6 and Nr4a1^−/− mice by quantitative PCR (Supplemental Fig. 2D). Consistent with previous reports, expression of both Cdkn1a and Cdkn1c was repressed in the absence of Nr4a1.

FIGURE 3.

Nr4a1^−/− CD8⁺ T cells display altered proliferative behavior. (A) Frequency of BrdU⁺ cells of TCRβ⁺CD8⁺ cells and frequency of BrdU⁺ cells of TCRβ⁺CD8⁺CXCR3⁺CD44^hi cells found in the lymph nodes. (B) Frequency of CFSE⁺CD45.1 (WT) cells and CFSE⁺CD45.2(Nr4a1^−/−) cells 7 d postcotransfer into MHC1^−/− hosts. (C) siRNA knockdown of Irf4 in anti-CD3,CD28–activated CD8 single-positive lymphocytes isolated from B6 or Nr4a1^−/− mice and labeled with CFSE. Data are representative of two separate experiments with at least three mice/group. *p < 0.05, **p < 0.005, unpaired two-tailed t test.

We further examined the expansion of Nr4a1-deficient CD8 T cells upon Ag-specific activation by transferring congenically mismatched CFSE-labeled CD8⁺ T cells isolated from the lymph nodes of OT1 and Nr4a1^−/− OT1 mice into B6 hosts (at a 1:1 ratio) and administering OVA 24 h later. Three days later, we found that the Nr4a1-deficient CD8 T cells expanded at a faster rate than the B6 OT1 control CD8⁺ T cells (Supplemental Fig. 2C).

Next, we adoptively cotransferred CFSE-labeled CD8⁺ T cells from lymph nodes of B6(CD45.1) and Nr4a1^−/−(CD45.2) mice into KbDb^−/− recipients. KbDb^−/− mice express no detectable classical MHC class I region–associated (Ia) H chains, although β2-microglobulin and the nonclassical class Ib proteins are expressed normally (18). At 7 d posttransfer, we analyzed the lymph nodes of the recipient mice for B6 (CD45.1⁺CFSE⁺) and Nr4a1^−/− (CD45.2⁺CFSE⁺) cells (Fig. 3B). B6 cells were found at an extremely low frequency, whereas abundant Nr4a1^−/− cells were found throughout the peripheral lymph nodes.

These data demonstrate that the absence of Nr4a1 promotes increased proliferation and expansion of the CD8 T cells that develop in Nr4a1^−/− mice as a result of the loss of repression of Irf4.

We went on to analyze whether the increase in Irf4 expression in Nr4a1^−/− CD8⁺ T cells directly accounts for the reported increased expansion. We first analyzed proliferation of CD8⁺ T cells expressing high versus low levels of IRF4 (Supplemental Fig. 2E) and found that CD8⁺ T cells that express higher levels of IRF4 exhibit an increase in proliferation based on Ki67 staining. We went on to knock down Irf4 expression through siRNA and analyzed proliferation of CD8⁺ T cells by CFSE dilution (Fig. 3C, Supplemental Fig. 1F) and Ki67 expression (Supplemental Fig. 2F). We found that, upon knockdown of Irf4 in both B6 and Nr4a1^−/− CD8⁺ T cells, proliferation (based on CFSE dilution) was significantly decreased. Analysis of Ki67 expression mirrored this data, again showing a lower proliferative rate upon knockdown of Irf4. Therefore, we directly demonstrated that the induction of IRF4 due to the absence of Nr4a1 results in the increased proliferation and expansion of Nr4a1-deficient CD8⁺ T cells.

Nr4a1 deficiency improves control of L. monocytogenes infection and increases generation of pathogen-specific effector CD8 T cells

Ectopic expression of IRF4 was shown to promote IFN-γ production by activated CD8⁺ T cells (9). We tested whether increased expression of IRF4 in Nr4a1-deficient CD8 T cells promotes IFN-γ production. In response to ex vivo stimulation with PMA and ionomycin, we found that a large proportion of Nr4a1^−/− CD8⁺ T cells produce IFN-γ compared with control B6 CD8⁺ T cells (Fig. 4A). Furthermore, knockdown of Irf4 by siRNA in OT1 and Nr4a1^−/−OT1 CD8 T cells resulted in reduced IFN-γ upon OVA activation in vitro (Supplemental Fig. 2H).

FIGURE 4.

Nr4a1 deficiency improves control of L. monocytogenes infection and increases generation of pathogen-specific effector CD8 T cells. (A) Frequency of IFNγ⁺ cells of total TCRβ⁺CD8⁺ cells in the lymph nodes upon PMA and ionomycin treatment. (B) B6, Nr4a1^−/−, and B6 +Nr4a1^−/− CD8 T cells were infected with L. monocytogenes, and CFU in the liver were determined at day 4 p.i. Each mark represents an individual mouse; horizontal lines denote the mean. (C) Total CD3⁺CD8⁺ T cells in the spleen of B6 and Nr4a1^−/− mice infected with L. monocytogenes at day 7 p.i. (D) H-2k^bOVA tetramer⁺ CD8⁺ T cells in the spleen of uninfected control, B6, and Nr4a1^−/− mice at day 7 p.i. (E) KLGR1 and IL-7Rα expression on H-2k^bOVA tetramer⁺ CD8⁺ T cells in the spleen of uninfected control, B6, and Nr4a1^−/− mice at day 7 p.i. (F) CCR7 and CD62L expression on H-2k^bOVA tetramer⁺ CD8⁺ T cells in the spleen of uninfected control, B6, and Nr4a1^−/− mice at day 7 p.i. (G) Granzyme B expression in H-2k^bOVA tetramer⁺ CD8⁺ T cells in the spleen of OT1 and OT1 Nr4a1^−/− mice at day 5 p.i. Data are representative of at least two separate experiments with at least four mice/group. *p < 0.05, **p < 0.005, unpaired two-tailed t test.

We hypothesized that upregulation of IFN-γ would give Nr4a1-deficient CD8⁺ T cells an advantage in clearing L. monocytogenes post in vivo infection. Infection of mice with L. monocytogenes induces a robust effector CD8⁺ T cell response that is crucial for clearance of the bacteria. To elucidate the role of Nr4a1 and the upregulation of IRF4, we infected B6 and Nr4a1^−/− mice, as well as B6 mice that received Nr4a1^−/− CD8⁺ T cells, with L. monocytogenes. Compared with B6 mice, both Nr4a1^−/− mice and B6 mice that received Nr4a1^−/− CD8 T cells exhibited far more clearance of the bacteria (Fig. 4B). To evaluate the CD8⁺ T cell response in Nr4a1^−/− mice postinfection (p.i.), we analyzed expansion of the total CD8⁺ T cell population and expansion of the Ag-specific CD8 population 7 d p.i. (Fig. 4C, 4D, Supplemental Fig. 1G). We also analyzed IRF4 expression in Ag-specific CD8 T cells p.i. (Supplemental Fig. 2I). Consistent with our previous data, polyclonal and Ag-specific Nr4a1-deficient CD8 T cells expanded at a faster rate than did the control CD8⁺ T cells. In response to acute infections, CD8⁺ T cells undergo clonal expansion and differentiation to short-lived effector cells (KLRG1^hiCD127^lo) and memory-precursor effector cells (KLRG1^loCD127^hi) (19). Examination of these populations revealed that Nr4a1 regulates the development of Ag-specific effector CD8 T cells and that, in the absence of Nr4a1, there is a significant increase in the development of effector CD8⁺ T cells (Fig. 4E). We expanded our examination to CD8⁺ memory T cells. Central memory cells are CCR7⁺CD62L^hi, whereas effector memory cells are CCR7⁻CD62L^lo. We found that, in the absence of Nr4a1, the effector memory population develops and expands at a faster rate (Fig. 4F). We further examined the functional difference in Nr4a1-deficient CD8 T cells through analysis of granzyme B expression 5 d p.i. with the L. monocytogenes strain recombinant for OVA. Consistent with our previous data demonstrating increased bacterial clearance (Fig. 4B), we found an increase in the frequency of granzyme B⁺ Nr4a1^−/− CD8 T cells (Fig. 4G, Supplemental Fig. 1H). Our data clearly demonstrate that the functional effects of Nr4a1 deficiency in effector CD8⁺ T cells is due to the resulting increase in the expression of IRF4. Therefore, our data demonstrate a crucial role that Nr4a1 plays in regulating IRF4 expression and, thereby, the development, expansion, and function of effector CD8⁺ T cells.

Disclosures

The authors have no financial conflicts of interest.