The transcription factor Interferon Regulatory Factor 4 is required for ...

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The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells Friederike Raczkowskia,1, Josephine Ritterb,1, Kira Heescha, Valéa Schumachera, Anna Guralnikb, Lena Höckerb, Hartmann Raiferb, Matthias Kleinc, Tobias Boppc, Hani Harbd, Dörthe A. Kesperd, Petra I. Pfefferled, Melanie Grusdate, Philipp A. Lange, Hans-Willi Mittrückera,2,3, and Magdalena Huberb,2,3 a Institute for Immunology, University Medical Center Hamburg–Eppendorf, 20246 Hamburg, Germany; bInstitute for Medical Microbiology and Hospital Hygiene, University of Marburg, 35033 Marburg, Germany; cInstitute for Immunology, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany; dInstitute for Laboratory Medicine and Pathobiochemistry, University of Marburg, 35043 Marburg, Germany; and e Department of Gastroenterology, Hepatology, and Infectious Diseases, Heinrich Heine University, 40225 Düsseldorf, Germany

Robust cytotoxic CD8+ T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8+ T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4−/−) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenesspecific CD8+ T cells with impaired effector phenotype and function. Transfer of wild-type CD8+ T cells into Irf4−/− mice improved bacterial clearance, suggesting an intrinsic defect of CD8+ T cells in Irf4−/− mice. Following transfer into wild-type recipients, Irf4−/− CD8+ T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4−/− CD8+ T cells rescued the defect. During acute infection, Irf4−/− CD8+ T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4−/− CD8+ T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4−/− CD8+ T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4+ T-cell differentiation, the identification of its decisive role in peripheral CD8+ T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.

ollowing infection with intracellular pathogens, specific CD8+ T cells become activated, proliferate, and differentiate into cytotoxic T cells, which are critical for the clearance of infection. Upon antigen encounter, these effector cells produce inflammatory cytokines and have the capability to kill infected cells. After resolution of infection, the bulk of effector cells dies; however, a small fraction remains as long-lived memory T cells that respond with rapid conversion into effector cells upon reexposure to the cognate pathogen (1). Phenotypic and functional markers allow distinction between short-lived effector CD8+ T cells and cells that give rise to longlived memory cells already at early stages of the response. Effector cells display a CD44hiCD62LloKLRG1hiIL7-Rαlo phenotype and memory precursor cells can be defined as CD44hiKLRG1loIL7Rαhi cells (1). Differentiation of CD8+ T cells into effector and memory cells is regulated by balanced expression of several transcription factors (TF). Whereas BCL-6 (2, 3), Eomesodermin (Eomes) (4), Id3 (5, 6), and TCF-1 (7) are associated with memory cell differentiation and longevity of cells, T-bet (encoded by Tbx21) (4, 8), Id2 (9), and Blimp-1 (encoded by Prdm1) (10, 11) promote effector cell development.

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The transcription factor IFN regulatory factor 4 (IRF4) controls class-switch recombination, germinal center B-cell formation, and plasma cell development (12). In CD4+ T cells, IRF4 is crucial for the differentiation into T helper (Th) subsets such as Th2, Th9, Th17, and Tfh cells (13–18). Mechanistically, IRF4 controls B-cell and dendritic cell differentiation by cooperative DNA binding with TFs of the Ets family on Ets-IRF composite elements (EICE) as well as by cooperation with basic leucine zipper transcription factor ATF-like (BATF)-JUN heterodimers in binding to AP-1IRF4 composite elements (AICE) (19–23). In contrast, differentiation of CD4+ T cells relies mainly on IRF4 binding to AICE elements (19, 21, 22). Moreover, there is evidence for cooperation of IRF4 with other TFs, including members of the NFAT, STAT, or homeobox protein families (12). There is only limited information on the function of IRF4 in CD8+ T cells. IRF4-deficient (Irf4−/−) mice are impaired in their response to lymphocytic choriomeningitis virus infection (24) and IRF4 appears to control expression of Eomes in these cells (25, 26). Here, we investigate the role of IRF4 in CD8+ T cells during an immune response against the intracellular bacterium Listeria monocytogenes and demonstrate an intrinsic role for IRF4 in the differentiation of peripheral cytotoxic T lymphocytes. Results IRF4 Is Essential for Clearance of L. monocytogenes. Infection of mice with L. monocytogenes induces a robust effector CD8+ T-cell response, which is crucial for clearance of bacteria (27). To elucidate the role of IRF4 in generation of protective CD8+ T cells, Irf4−/− and WT mice were infected with L. monocytogenes. Compared with WT mice, Irf4−/− mice were compromised in the eradication of L. monocytogenes (Fig. 1A and Fig. S1A). Furthermore, the expansion of the CD8+ T-cell population and the acquisition of the CD62LloCD44hiKLRG1hi effector phenotype by CD8+ T cells were greatly impaired in Irf4−/− mice (Fig. S1 B– F). However, we observed an increase in the proportion of CD44hi cells, indicating activation of Irf4−/− CD8+ T cells during infection (Fig. S1D). To evaluate whether the impaired clearance of

Author contributions: F.R., J.R., T.B., P.A.L., H.-W.M., and M.H. designed research; F.R., J.R., K.H., V.S., A.G., L.H., H.R., M.K., H.H., D.A.K., P.I.P., M.G., H.-W.M., and M.H. performed research; F.R., J.R., H.-W.M., and M.H. analyzed data; and F.R., H.-W.M., and M.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

F.R. and J.R. contributed equally to this work and are listed in alphabetical order.

2

H.-W.M. and M.H. contributed equally to this work.

3

To whom correspondence may be addressed. E-mail: [email protected] or [email protected]

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1309378110/-/DCSupplemental.

PNAS | September 10, 2013 | vol. 110 | no. 37 | 15019–15024

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Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, ON, Canada, and approved July 29, 2013 (received for review May 20, 2013)

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Fig. 1. Impaired control of L. monocytogenes infection by IRF4-deficient CD8+ T cells. (A) Irf4−/− and WT mice were infected with L. monocytogenes and colony forming units (CFU) in livers were determined at indicated days p.i. (B) Irf4−/− mice were reconstituted with Ly5.1+ congenic WT CD8+ T cells and infected with L. monocytogenes. CFU in livers were determined at day 12 p.i. (C) CD44, CD62L, and KLRG1 expression by splenic CD8+ T cells (day 12 p.i.) in reconstituted Irf4−/− (Ly5.1−) mice. (A and B) CFU for individual mice and the median of combined results from two independent experiments are shown (n = 12–14). The dashed line gives the detection limit. (C) Numbers give percentage of positive cells. Experiments were repeated twice with consistent results.

L. monocytogenes was caused by defective function of CD8+ T cells, we transferred WT CD8+ T cells into Irf4−/− mice. At day 12 postinfection (p.i.), significantly lower bacterial numbers were found in Irf4−/− mice after transfer of WT CD8+ T cells and in contrast to Irf4−/− CD8+ T cells, a substantial proportion of transferred WT cells displayed a CD62LloKLRG1hi effector phenotype (Fig. 1 B and C and Fig. S1G). Thus, WT CD8+ T cells acquired effector properties in an IRF4-deficient environment, indicating that intrinsic defects in CD8+ T cells were at least in part responsible for the impaired clearance of L. monocytogenes by Irf4−/− mice. Impaired Pathogen-Specific Effector CD8+ T-Cell Response in Irf4−/− Mice. To characterize the function of IRF4 in an antigen-specific

setting, WT and Irf4−/− mice were infected with an L. monocytogenes strain recombinant for chicken ovalbumin (LmOVA). Irf4−/− mice also failed to clear the LmOVA infection (Fig. S2A). We noted a significant reduction in OVA-specific CD8+ T cells in tissues of Irf4−/− mice and these cells failed to acquire a CD62LloKLRG1hi phenotype (Fig. 2 A–C). OVA-specific cytokine production was also greatly impaired in Irf4−/− mice compared with WT mice (Fig. 2D). Infection with an L. monocytogenes strain recombinant for gp33 from LCMV revealed a comparable defect (Fig. S2 B–E). Furthermore, we failed to detect substantial responses to H2-M3–restricted formyl-methionin (f-met) peptides of L. monocytogenes (Fig. S2 B and C). Thus, Irf4−/− mice fail to mount a regular CD8+ effector response to several immunodominant peptides presented by different MHC molecules during infection with L. monocytogenes. Irf4−/− CD8+ T Cells Display Altered Proliferative Behavior. The anal-

ysis of L. monocytogenes clearance suggested an intrinsic defect of CD8+ T cells in Irf4−/− mice (Fig. 1 B and C). To fully isolate the IRF4 deficiency to CD8+ T cells, we conducted competitive transfers of small numbers of both WT and Irf4−/− OVA-specific OT-I CD8+ T cells into congenic WT recipients, followed by LmOVA infection. At day 3 after transfer and infection, we found similar numbers of WT and Irf4−/− OT-I cells in spleens of recipient mice; however, at day 5 the ratio changed to approximately 10:1 (Fig. 3 A and B and Fig. S3 A and B). Reduced accumulation of Irf4−/− OT-I cells was also found in other tissues (Fig. S3C), and noncompetitive T-cell transfers gave similar results (Fig. S4 A and B). Phenotypically, transferred Irf4−/− OT-I cells displayed less pronounced up-regulation of 15020 | www.pnas.org/cgi/doi/10.1073/pnas.1309378110

CD44, CXCR3, and CD25, suggesting that they reacted to LmOVA, although to a lesser extent than WT cells. Furthermore, they failed to down-regulate CD62L and CD27 and to up-regulate KLRG1, again confirming that phenotypic alterations of CD8+ T cells observed in Irf4−/− mice were due to an intrinsic defect of these cells (Fig. 3C and Fig. S3 D and G). Consistently, IRF4 was rapidly induced by polyclonal or antigen-specific stimulation and during L. monocytogenes infection, its induction correlated with the acquisition of the effector phenotype by CD8+ T cells (Fig. S5 A–F). To elucidate whether reduced accumulation of Irf4−/− CD8+ T cells was caused by restricted proliferation, we measured carboxyfluorescein succinimidyl ester (CFSE) dilution. Both WT and Irf4−/− OT-I cells had proliferated extensively as measured at day 5 posttransfer (Fig. 3D). Because impaired accumulation of Irf4−/− OT-I cells was evident at day 5 but not at day 3 posttransfer and infection, we speculated that Irf4−/− CD8+ T cells might not maintain an initial proliferation. Indeed, BrdU incorporation by Irf4−/− cells between days 4 and 5 was significantly lower, compared with that by WT cells (Fig. 3E), suggesting reduced proliferation of Irf4−/− cells at this stage of infection. The proliferative defect of Irf4−/− cells was also detectable in vitro and could not be rescued by the addition of high amounts of IL-2 (250 units) (Fig. S6 A and B). The rate of apoptosis was reduced in Irf4−/− OT-I cells at day 5 p.i. as determined by fluorescent labeled inhibitor of caspases (FLICA) and Annexin V staining, and Irf4−/− cells expressed higher levels of the prosurvival factor Bcl2 (Fig. 3F and Fig. S3 E and F), reflecting again their defect to mature into effector cells that are more prone to apoptosis (28). Furthermore, the expression of the exhaustion markers CD244, LAG-3, and PD-1 (29) was not enhanced in Irf4−/− OT-I cells compared with WT cells at day 5 of infection (Fig. S3G). Thus, reduced numbers of Irf4−/− CD8+ T cells during acute L. monocytogenes infection were most likely caused by an intrinsic failure to maintain proliferation and not due to increased apoptosis or exhaustion.

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Fig. 2. IRF4 deficiency impairs generation of pathogen-specific effector CD8+ T cells. (A–D) Irf4−/− and WT mice were infected with LmOVA and analyzed at day 12 p.i. (A) H-2KbOVA257–264-dextramer staining of CD8+ T cells isolated from spleens. (B) Numbers of H-2KbOVA257–264-dextramer+ CD8+ T cells in spleens (SPL), livers (LV), and bone marrow (BM) (mean ± SEM, n = 4). (C) CD44 and CD62L or KLRG1 and IL-7Rα expression on H-2KbOVA257–264dextramer+ CD8+ T cells isolated from spleens of WT and Irf4−/− mice. (D) Spleen cells from Irf4−/− and WT were stimulated with OVA257–264 peptide and analyzed on a CD8+ gate for intracellular IFN-γ, TNF-α, and IL-2. Bar graphs display percentage values of IFN-γ+ and IFN-γ+TNF-α+ CD8+ T cells (mean ± SEM, n = 4). (A, C, and D) Numbers give percentage of positive cells. Experiments were repeated twice with consistent results.

Raczkowski et al.

inflammatory cytokine production is central for the protective capacity of CD8+ T cells. To evaluate the role of IRF4 in CD8+ T cells in this process, we again used competitive transfer of WT and Irf4−/− OT-I cells. Although we detected similar accumulation of Irf4−/− and WT OT-I cells at day 3 posttransfer and infection, Irf4−/− cells already displayed impaired IFN-γ, TNF-α, and GzmB production at this time point and the defect in GzmB expression was even more pronounced at day 5 (Fig. 4 A, B, and D and Fig. S7 A and B). Irf4−/− cells also failed to produce IL-2 (Fig. 4C). The defect of Irf4−/− cells in production of IFN-γ and TNF-α was also detectable in vitro and could not be rescued by addition of high amounts of IL-2 (Fig. S6C). The mRNA analysis of sorted WT and Irf4−/− OT-I cells isolated from acute infection revealed decreased levels for the cytotoxic molecules GzmB, Granzyme K, and Perforin 1 (Fig. 4E). Consistent with this result, we detected impaired cytotoxicity of Irf4−/− OT-I cells in an in vivo kill assay after transfer of LmOVA-activated WT and Irf4−/− OT-I cells (Fig. 4F). Importantly, diminished cytotoxicity was not caused by loss of Irf4−/− OT-I cells because we detected similar numbers of transferred cells in recipients of WT and Irf4−/− cells (Fig. 4F). In summary, these results demonstrate an intrinsic defect of Irf4−/− CD8+ T cells to acquire functions of terminal effector cells during L. monocytogenes infection. To exclude developmental defects of Irf4−/− CD8+ T cells, we retrovirally overexpressed IRF4 in LmOVA-primed WT or Irf4−/− OT-I cells (7, 8). Cells were transferred into congenic mice, which were infected with LmOVA (Fig. 4 G and H). WT cells transduced with control virus (control-RV) or IRF4-expressing virus (IRF4RV) displayed similar production of IFN-γ and TNF-α. Irf4−/− OT-I cells transduced with control-RV barely produced cytokines. In contrast, Irf4−/− cells transduced with IRF4-RV produced IFN-γ and TNF-α. Thus, forced expression of IRF4 in Irf4−/− CD8+ T cells rescued at least partially the cytokine production, corroborating the crucial role of IRF4 for CD8+ effector differentiation and excluding developmental defects. IRF4 Controls Expression of Transcription Factors Regulating CD8+ T-Cell Fate Decision. Differentiation of effector CD8+ T cells is

controlled by coordinated expression of several TFs (2–11). Therefore, WT and Irf4−/− OT-I CD8+ T cells were sorted from recipient mice during acute L. monocytogenes infection and mRNA levels for different TFs were determined by quantitative RT-PCR analysis (Fig. 5). Consistent with severely impaired effector differentiation of Irf4−/− CD8+ T cells, we found diminished expression of TFs important for CD8+ effector-cell development such as Prdm1 (encoding Blimp-1), Id2, and Tbx21 (encoding T-bet) in these cells. Notably, the expression of TFs associated with memory T-cell differentiation such as BCL-6, Raczkowski et al.

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Eomes, and Id3 was increased in Irf4−/− CD8+ T cells. This feature combined with the CD44hiCD62Lhi memory-like phenotype of activated Irf4−/− CD8+ T cells (Fig. S1D and Fig. 3C) suggested that absence of IRF4 might promote the formation of memory CD8+ T cells. However, in two experimental approaches in which either CD8+ T cells were directly analyzed in Irf4−/− mice or Irf4−/− OT-I cells were analyzed after transfer into WT recipients, we noted decreased numbers of OVA-specific CD8+ T cells 40 d after LmOVA infection and these cells were profoundly impaired in IFN-γ and TNF-α production after stimulation (Fig. S8 A–H). Thus, the formation as well as the function of long-lived memory CD8+ T cells was markedly impaired in the absence of IRF4. IRF4 Binds Directly to Regulatory Elements of the Prdm1 Gene in CD8+ T Cells. Blimp-1–deficient CD8+ T cells display impaired cyto-

toxicity and express diminished levels of KLRG1 and Tbx21, whereas the expression of Eomes and Bcl6 is increased in these cells. Therefore, Blimp-1 has been defined as a central TF for terminal effector CD8+ T-cell differentiation (10, 11). Because of similarities in the phenotype of Irf4−/− CD8+ T cells and that described for Blimp-1–deficient CD8+ T cells and strong reduction of Prdm1 expression in Irf4−/− CD8+ T cells during acute L. monocytogenes infection, we hypothesized that IRF4 regulates Prdm1. To test this, CD8+ T cells from WT and Irf4−/− mice were activated in vitro and then cultured with cytokines contributing to CD8+ T-cell activation (Fig. 6A). Without addition of cytokines, Irf4−/− CD8+ T cells showed reduced expression of Prdm1 compared with WT cells. IL-2 even in high concentrations and IL-12 did not change the expression level of Prdm1 in both populations (Fig. 6A and Fig. S6D). Consistent with published data for CD4+ T cells (30), IL-21 strongly induced Prdm1 and Blimp-1 protein in WT CD8+ T cells, which corresponded to enhanced IRF4 levels. In agreement with our ex vivo data, Prdm1 expression was markedly lower in Irf4−/− cells and Blimp-1 was undetectable (Fig. 6 A–C). Reduced expression was not due to a failure of Irf4−/− CD8+ T cells to react to IL-21, because WT and Irf4−/− cells displayed similar phosphorylation of STAT3 upon IL21 treatment (Fig. S9A). Furthermore, the transduction of Irf4−/− CD8+ T cells with IRF4-expressing retrovirus induced enhanced expression of Prdm1 compared with transduction with control retrovirus, suggesting direct regulation of Blimp-1 by IRF4 (Fig. 6D and Fig. S9B). Previous studies (30) have identified an IL-21 response element downstream of Prdm1 that binds IRF4 in B cells and CD4+ T cells and is required for optimal Prdm1 expression. Our chromatin immunoprecipitation (ChIP) analysis revealed strong binding of IRF4 to this element after treatment of WT CD8+ T cells with IL-21. Computational analysis of the 5′ region of the Prdm1 gene locus revealed further putative IRF4-binding PNAS | September 10, 2013 | vol. 110 | no. 37 | 15021

IMMUNOLOGY

IRF4 Regulates CD8+ T-Cell Effector Development in a Cell-Intrinsic Manner. Acquisition of effector functions such as cytotoxicity and

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Fig. 3. IRF4 intrinsically regulates the phenotype d5 *** d3 5 20009 939 and proliferation of effector CD8+ T cells. Purified 1849 OT-I WT 8753 242 13897 OT-I Irf4-/CD8+ T cells from Irf4−/− (CD90.1−CD90.2+) OT-I mice 4 52.7 + + OT-I WT and WT OT-I mice (CD90.1 CD90.2 ) were mixed in 3 OT-I Irf4-/a ratio of 1:1 and labeled with CFSE. A total of 4 × 47.3 2 104 OT-I cells were injected into LmOVA-infected d3 CD44 d5 KLRG1 CD62L CD90.1+CD90.2− congenic mice. Transferred cells d5 from spleens of recipient mice were analyzed at d5 BrdU d4 d5 CFSE d0 d5 6 30 15 *** 92.4 6847 4367 210 indicated days p.i. (A) CD90.1 and CD90.2 staining 5 1288 1493 339 *** 20 4 of transferred OT-I cells at days 3 and 5 p.i. Numbers 10 ns. 7.2 3 give the percentage values for Irf4−/− and WT OT-I 10 5 2 −/− cells. (B) Total numbers of transferred Irf4 and 1 CD90.2 0 0 WT OT-I cells at indicated days p.i. (C) Expression of 0 BrdU d0 d5 d5 CFSE d0 d5 FLICA CD62L, CD44, and KLRG1 on WT and Irf4−/− OT-I −/− cells at day 5 p.i. (D) CFSE profiles of Irf4 and WT OT-I cells at day 5 p.i. [dotted line and shaded bar: CFSE staining of mixed cells before transfer (day 0)]. (E) Mice received BrdU at day 4 p.i. and were analyzed at day 5 p.i. Plots show BrdU incorporation by transferred cells. (F) FLICA binding by transferred Irf4−/− and WT cells before transfer (day 0) and at day 5 p.i. Numbers in all histograms give the mean fluorescence intensity (MFI). Bars give the mean ± SEM, n = 3–5. Experiments were repeated twice with consistent results.

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Fig. 4. IRF4 regulates effector CD8+ T-cell differentiation. (A–D) Purified CD8+ T cells from Irf4−/− and WT OT-I mice were mixed in a ratio of 1:1 and 4 × 104 cells were injected into LmOVA-infected congenic mice. Transferred cells from spleens of recipient mice were analyzed at day 3 and day 5 p.i. Bars give the mean ± SEM, n = 4. (A, C, and D) Cytokine production is shown after stimulation with OVA257–264 peptide. Numbers in plots and bars show percentages of positive cells. (B) Numbers in histograms and bars give the MFI of GzmB. (E) Irf4−/− and WT OT-1 cells were sorted on day 6 after transfer and LmOVA infection and mRNA expression levels were determined by qRT-PCR. Relative expression was calculated by setting the expression levels in Irf4−/− OT-I cells to 1 (mean ± SD of duplicate PCR samples). (F) After activation in LmOVA-infected recipient mice, equal numbers of purified Irf4−/− and WT OT-I cells were retransferred into congenic WT mice that received target and control cells. Graphs show percentage of killing and total numbers of OT-I cells recovered from spleens after 4 h (mean ± SEM, n = 3). (G and H) Activated WT (G) or Irf4−/− (H) OT-I cells were transduced with retrovirus encoding IRF4-GFP (IRF4-RV) or a control retrovirus encoding GFP (controlRV) and transferred into LmOVA-infected congenic mice. At day 8 p.i., spleen cells were stimulated with OVA257–264 peptide and GFP+ OT-I cells were analyzed. Numbers give mean ± SEM, n = 4, of IFN-γ+ and IFN-γ+ TNF-α+ cells. Experiments were repeated twice (A–F) or four times (G and H) with consistent results.

sites, for two of which we found specific binding (Fig. 6E). Precipitation with anti-IRF4 Ab resulted in significant enrichment of the analyzed regulatory elements of Prdm1 compared with precipitation with control IgG and there was no significant binding of IRF4 to control sequences from the 5′ region of the Rpl32 gene, which does not contain canonical IRF motifs (Fig. S9C). These results demonstrate that in CD8+ T cells, IRF4 is essential for Blimp-1 expression and binds specifically to regulatory regions of the Prdm1 gene and suggest that the regulation of Blimp-1 by IRF4 contributes to the impaired effector differentiation of Irf4−/− CD8+ T cells. Discussion Despite the established role for IRF4 in B-cell (12) and CD4 + T-cell differentiation (13–19, 21, 22), its function in CD8+ T cells during infection has not been closely evaluated. Here, we show that Irf4−/− mice mostly failed to eradicate the intracellular bacterium L. monocytogenes. This defect was caused largely by a failure in the generation of a protective CD8+ T-cell response. 15022 | www.pnas.org/cgi/doi/10.1073/pnas.1309378110

Characterization of CD8+ T cells in transfer assays revealed that Irf4−/− CD8+ T cells responded to infection with initial proliferation. Activated cells showed phenotypic changes and produced cytokines. However, Irf4−/− CD8+ T cells could not sustain proliferation and retained a “precursor-like” state, as indicated by diminished cytokine response, low expression of cytotoxic proteins, and defective cytotoxicity. This deficiency in maturation into fully functional effector cells was CD8+ T-cell intrinsic and could be rescued by retroviral IRF4 expression in Irf4−/− CD8+ T cells. The retroviral expression studies also exclude developmental defects, such as altered thymic maturation, as a main cause of impaired CD8+ T-cell responses in Irf4−/− mice. This result is consistent with our results and a recent publication (25), which demonstrate normal thymic T-cell maturation in Irf4−/− mice. The impaired maturation of Irf4−/− CD8+ T cells during acute infection was accompanied by decreased expression levels of TFs central to the formation of effector cells such as Blimp-1, Id2, and T-bet (1), suggesting that IRF4 controls their expression. Because Irf4−/− CD8+ T cells resemble Blimp-1–deficient CD8+ T cells with regard to impaired cytotoxicity and diminished KLRG1 expression (10, 11), we hypothesized that IRF4 functions upstream of Blimp-1 in regulating effector-cell maturation. Indeed, we could demonstrate that Blimp-1 expression was reduced in Irf4−/− CD8+ T cells. Conversely, the overexpression of IRF4 in Irf4−/− CD8+ T cells enhanced Prdm1 expression. Reduced Prdm1 level was also evident in Irf4−/− CD8+ T cells after addition of high amounts of IL-2. These results point to a direct control of Prdm1 by IRF4. Accordingly, IRF4 specifically bound to the IL-21 responsive element and other regulatory regions of the Prdm1 gene. Recently, IRF4 was shown to be crucial in IL21–induced Blimp-1 expression in B cells and CD4+ T cells (30) as well as in Treg cells (31). Our data extend these observations to CD8+ T cells and suggest the IRF4–Blimp-1 axis as a common regulatory mechanism in lymphocytes. Because Blimp-1 controls terminal effector differentiation and the expression of other TFs involved in lymphocyte differentiation such as T-bet, Eomes, BCL-6, and Id3 (10, 11), impaired induction of Blimp-1 in the absence of IRF4 could be largely responsible for the defective effector differentiation of Irf4−/− CD8+ T cells. To prove this concept, we tried to overexpress Blimp-1 in Irf4−/− CD8+ T cells. However, due to the large size of Blimp-1, we were not able to reliably express Blimp-1 in CD8+ T cells. Furthermore, Blimp-1–deficient CD8+ T cells differ from Irf4−/− CD8+ T cells in IL-2 production and memory cell formation (10, 11); thus, it is likely that IRF4 influences CD8+ T-cell differentiation by additional Blimp-1–independent mechanisms. It is likely that similarly to that in CD4+ T cells, cooperative binding of IRF4 with BATF-JUN heterodimers also occurs in CD8+ T cells. BATF-deficient mice show impaired effector CD8+ T-cell differentiation, characterized by reduced expression of IFN-γ, Perforin, and T-bet, which was caused by changes in

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Fig. 5. IRF4 balances the expression of transcription factors controlling CD8+ T-cell differentiation. Purified CD8+ T cells (4 × 104) from Irf4−/− and WT OT-I mice were individually transferred into LmOVA-infected congenic WT recipients. After 6 d, OT-I cells were sorted and analyzed by qRT-PCR. mRNA levels were normalized to Hprt1 and relative expression was calculated by setting of the lowest experimental value to 1. Bars give the mean (± SD) of duplicate PCR samples. The experiment was repeated twice with consistent results.

Raczkowski et al.

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Fig. 6. IRF4 controls Prdm1 expression in CD8+ T cells. (A) Purified CD8+ T cells from Irf4−/− and WT OT-I mice were preactivated with immobilized antiCD3 mAb plus soluble anti-CD28 mAb and IL-2 for 3 d and then rested for 24 h. Prdm1 mRNA was determined by qRT-PCR at 6 and 24 h after treatment as indicated. Bars give the mean (± SD) of duplicate PCR samples. (B and C) Preactivated Irf4−/− and WT CD8+ T cells remained untreated or were treated with IL-21 for 24 h and Blimp-1 (B), IRF4 (C), or β-Actin levels were determined by immunoblotting. (D) Purified CD8+ T cells from Irf4−/− OT-I mice were preactivated as in A and transduced with retrovirus encoding IRF4 (IRF4-RV) or control retrovirus (Ctrl-RV). After 3 d, cells were rested for 24 h and then stimulated with IL-21. After 24 h, Prdm1 mRNA was determined by qRT-PCR. Bars give the mean (± SD) of duplicate PCR samples. (A–D) The experiments were repeated two times with consistent results. (E) ChIP of IRF4 was performed with preactivated Irf4−/− and WT CD8+ T cells without stimulation or treated with IL-21 for 1 h. Input DNA and precipitated DNA were quantified by qRT-PCR with primer pairs specific for regulatory regions of Prdm1; the same chromatin was used for control ChIP experiments with control IgG. Precipitated DNA is presented relative to input (% of input). Values for nonspecific binding (as determined by using control IgG) were subtracted; nd, not detectable. Shown is mean ± SEM of combined results from three independent experiments (n = 9); ns, not significant.

epigenetic remodeling and energy metabolism (32). It is possible that BATF and IRF4 cooperatively affect CD8+ T-cell effector differentiation either by regulation of effector proteins and TFs or by promoting changes in metabolic pathways. In B cells and CD4+ T cells, IRF4 additionally cooperates with several other TFs, including members of the ETS family, E47, NFATc2, STAT3, and STAT6 (12). In CD8+ T cells IRF4 might interact with some of these molecules as well to influence differentiation. Analysis of TFs in Irf4−/− CD8+ T cells revealed up-regulation of BCL-6, Eomes, and Id3. We and others have recently described high Eomes expression in Irf4−/− CD8+ T cells (25, 26). High expression of BCL-6, Eomes and Id3 and low expression of Blimp-1, Id2, and T-bet are associated with the development of memory CD8+ T cells (1). However, only marginal numbers of LmOVA-specific CD8+ T cells were detectable in Irf4−/− mice and they mostly failed to produce cytokines upon stimulation. Thus, IRF4 controls maturation processes essential for the generation of both effector and memory CD8+ T cells. The rapid induction of IRF4 after T-cell activation suggests that IRF4 acts Raczkowski et al.

already at early differentiation steps that are common to both fates. Along with this model, IRF4 may function as a permissive factor by rendering precursor-like cells responsive to fate-shaping environmental cues such as cytokines, costimulatory signals, or different antigen loads that are encountered in the course of infection. Consequently, loss of IRF4 causes a disbalanced transcriptional program that immediately affects effector-cell generation during acute infection but also prevents the formation of functional memory CD8+ T cells. In conclusion, our results identify IRF4 as a central regulator of peripheral CD8+ T-cell differentiation. These data parallel observations in late stages of B-cell development (12) and in generation of Th-cell subsets (13–18) and thus support the idea of conserved transcriptional modules regulating peripheral differentiation of adaptive lymphocytes (33). Mice and L. monocytogenes Infection. C57BL/6, congenic CD45.1 (Ly5.1) (6.SJLPtprca Pep3b/BoyJ; The Jackson Laboratory), congenic CD90.1 (B6.PL-Thy1a/ CyJ; The Jackson Laboratory), OT-I, (OT-I×CD90.1)F1, Irf4−/−, and Irf4−/− OT-I mice were bred at the animal facilities of the University Medical Center Hamburg–Eppendorf or of the Biomedical Research Center, University of Marburg. Experiments were conducted according to the German animal protection law. Mice were infected i.p. with 5 × 104 colony-forming units (cfu) of the WT L. monocytogenes strain EGD (Lm) or with 105 cfu of L. monocytogenes strains recombinant for ovalbumin (LmOVA) or for gp33 of LCMV (LmGP33) (34). Bacterial titers were quantified as previously described (35). Cell Transfer Experiments. CD8+ T cells from spleens of WT or Irf4−/− OT-I mice were purified by negative magnetic activated cell sorting (MACS) selection (Miltenyi Biotec) according to the manufacturer’s protocol. For noncompetitive transfers, 4 × 104 WT or Irf4−/− OT-I CD8+ T cells were injected i.v. into Ly5.1+ or CD90.1+ congenic mice. For competitive transfers, a total of 4 × 104 WT OT-I (CD90.1+ CD90.2+) and Irf4−/− OT-I (CD90.1− CD90.2+) cells in ratio 1:1 were injected i.v. into congenic mice (CD90.1+ CD90.2−). In some experiments, cells were stained with 2 μM of CFSE (Molecular Probes). Recipient mice were infected with 105 cfu LmOVA. For reconstitution experiments, MACS-purified WT Ly5.1+ CD8+ T cells were injected into Irf4−/− recipients (5 × 106 per mouse) 1 d before L. monocytogenes infection. Isolation of Cells and Flow Cytometry. Lymphocytes were isolated from different tissues as previously described (36). For extracellular staining, cells were incubated with rat serum and anti-CD16/CD32 mAb and then stained with specific mAb as indicated. Ovalbumin-specific CD8+ T cells were detected with H-2KbOva257–264 dextramers (Immudex). The cells were analyzed by flow cytometry, using a FACS Canto II, a FACSCalibur, or an Aria III (Becton-Dickinson). Results were analyzed with the DIVA software (BectonDickinson) or the FlowJo Software (Tree Star). Fluorochrome-conjugated mAbs to CD3 (eBio500A2 or 145-2C11), CD4 (RM4-5), CD8α (53-6.7), CD25 (PC61.5), CD44 (IM7), Ly5.1 (A20), CD62L (MEL-14), CD90.1 (His51), CD90.2 (53-2.1), CD127/IL-7Rα (A7R34), CD244 (2B4), TCR-Vα2 (B20.1), CXCR3 (CXCR3-173), KLRG1 (2F1), LAG-3 (C9B7W), PD-1 (J43), Granzyme B (GB12), IRF4 (3E4), Bcl2 (10C4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), and IL-2 (JES65H4) were purchased from BioLegend, eBioscience, or BD Pharmingen. Flow cytometric sorting was performed on an Aria III. Flow Cytometry-Based Assays. For the characterization of cytokine production, cells were incubated with 10−6 M of the peptides OVA257–264 (SIINFEKL) or LCMV gp33–41 (KAVYNFATM) or of a mixture of the L. monocytogenes f-met peptides fMIGWII, fMIVIL, and fMIVTLF (all JPT) in the presence of brefeldin A (Sigma Aldrich) for 4–5 h. Cells were stained extracellularly, fixed, and stained intracellularly. Intracellular staining for GzmB and IRF4 was done without prior stimulation. Anti-BrdU mAbs (BD Pharmingen) were used according to the manufacturer’s protocols. Mice received 1 mg BrdU i.p. 1 d before analysis. Apoptosis was determined with the FLICA Kit (Immunochemistry Technologies) according to the manufacturer’s protocol. Dead cells were identified by DAPI staining. Quantitative Real-Time PCR. A total of 4 × 104 WT or Irf4−/− OT-I CD8+ T cells were transferred into congenic WT mice, which were infected with LmOVA. Six days p.i., OT-I cells were FACS-sorted from spleens and total RNA was

PNAS | September 10, 2013 | vol. 110 | no. 37 | 15023

IMMUNOLOGY

8

Relative expression

Relative mRNA expression

A

prepared using the RNeasy Micro Kit (Qiagen). cDNA synthesis and PCR were performed as described previously (37). mRNA expression levels were normalized to hypoxanthine–guanine phosphoribosyl transferase (Hprt1) expression and relative fold differences were calculated. The lowest experimental value was set to 1. The primer sets have been described previously (37). The primer pair for Bcl6 detection was forward, 5′-CCTGTGAAATCTGTGGCACTCG3′, and reverse, 5′-CGCAGTTGGCTTTTGTGACG-3′. Immunoblotting. Whole-cell lysates were prepared from purified CD8+ T cells without stimulation or after in vitro stimulation. Immunoblotting was performed, as described previously (26). Briefly, proteins were fractionated by SDS/ PAGE, transferred to nitrocellulose membrane, immunoblotted with pSTAT3 Tyr705 (9131; Cell Signaling Technology), IRF4 (M-17; sc6059; Santa Cruz) or Blimp-1 (Novus) antibodies, and then reprobed with antibodies to total STAT3 (124H6; 9139; Cell Signaling Technology) or β-Actin (Sigma-Aldrich). Chromatin Immunoprecipitation Assays. For ChIP experiments, CD8+ T cells enriched by negative MACS selection were preactivated with plate-bound anti-CD3 mAb (2 μg/mL) and soluble anti-CD28 mAb (1 μg/mL) in the presence of rhIL-2 (50 units/mL) for 3 d, rested overnight, and stimulated with IL-21 (100 ng/mL) for 1 h. A total of 2 × 106 cells were fixed with 1% formaldehyde for 10 min at room temperature to preserve the protein–DNA interactions. Subsequently, ChIP was performed as described previously (38) with antibodies against IRF4 (M-17; Santa Cruz). Quantitative RT-PCR with the precipitated chromatin was performed to calculate the percentage of input. Primer sequences are provided in Table S1. All amplifications were performed in triplicate with SYBR Green PCR Master Mix (Qiagen). Control ChIP was performed with a respective isotype control antibody to ensure specificity. After normalization of the data according to the isotype control, the specific pulldown (percentage of input chromatin) was calculated.

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15024 | www.pnas.org/cgi/doi/10.1073/pnas.1309378110

In Vivo Cytotoxicity Assay. Single transfers of CD8+ T cells from WT OT-I mice or Irf4−/− OT-I mice into congenic mice and LmOVA infection were done. On day 5 p.i., WT or Irf4−/− OT-I CD8+ T cells were recovered from spleens of recipient mice by MACS purification and equalized numbers of cells (1 × 106) were transferred into naive mice. One day later, target and control cells were prepared by loading of spleen cells with 10−6 M of the peptides OVA257–264 (target cells) or LCMV gp33–41 (control cells), which were then stained with 2 μM or 0.2 μM CFSE, respectively. Target and control cells were then mixed in a 1:1 ratio and a total of 6 × 106 cells were injected into the recipients of activated OT-I cells. Four hours later, spleen cells were isolated and killing as well as OT-I CD8+ T-cell numbers were determined. Killing was calculated as follows:  . % Killing = 100 − CFSEhi sample=CFSElo sample    CFSEhi control=CFSElo control × 100 :

Statistics. All described experiments were performed at least two times with similar results. For statistical analysis of frequencies and cell numbers, we applied an unpaired two-tailed Student’s t test. Bacterial titers were compared using the Mann–Whitney test. P values are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Additional methods can be found in SI Material and Methods. ACKNOWLEDGMENTS. The authors thank Dr. Hao Shen for providing recombinant L. monocytogenes strains and Dr. Timo Lischke and Bärbel Camara for technical advice and assistance. This work was supported by the Deutsche Forschungsgemeinschaft through grants to M.H. (HU 1824/21) and H.-W.M. (MI 476/3, SFB841, and KFO228) as well as to P.I.P. (SFB/TR22).

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