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• W2773141414 abstract "•Ablation of IRF4 induces transplant acceptance by establishing T cell dysfunction•IRF4 represses PD-1, Helios, and other molecules associated with T cell dysfunction•Irf4‒/‒ T cell dysfunction is initially reversible but later becomes irreversible•Trametinib inhibits IRF4, abrogates EAE development, and prolongs allograft survival CD4+ T cells orchestrate immune responses and destruction of allogeneic organ transplants, but how this process is regulated on a transcriptional level remains unclear. Here, we demonstrated that interferon regulatory factor 4 (IRF4) was a key transcriptional determinant controlling T cell responses during transplantation. IRF4 deletion in mice resulted in progressive establishment of CD4+ T cell dysfunction and long-term allograft survival. Mechanistically, IRF4 repressed PD-1, Helios, and other molecules associated with T cell dysfunction. In the absence of IRF4, chromatin accessibility and binding of Helios at PD-1 cis-regulatory elements were increased, resulting in enhanced PD-1 expression and CD4+ T cell dysfunction. The dysfunctional state of Irf4-deficient T cells was initially reversible by PD-1 ligand blockade, but it progressively developed into an irreversible state. Hence, IRF4 controls a core regulatory circuit of CD4+ T cell dysfunction, and targeting IRF4 represents a potential therapeutic strategy for achieving transplant acceptance. CD4+ T cells orchestrate immune responses and destruction of allogeneic organ transplants, but how this process is regulated on a transcriptional level remains unclear. Here, we demonstrated that interferon regulatory factor 4 (IRF4) was a key transcriptional determinant controlling T cell responses during transplantation. IRF4 deletion in mice resulted in progressive establishment of CD4+ T cell dysfunction and long-term allograft survival. Mechanistically, IRF4 repressed PD-1, Helios, and other molecules associated with T cell dysfunction. In the absence of IRF4, chromatin accessibility and binding of Helios at PD-1 cis-regulatory elements were increased, resulting in enhanced PD-1 expression and CD4+ T cell dysfunction. The dysfunctional state of Irf4-deficient T cells was initially reversible by PD-1 ligand blockade, but it progressively developed into an irreversible state. Hence, IRF4 controls a core regulatory circuit of CD4+ T cell dysfunction, and targeting IRF4 represents a potential therapeutic strategy for achieving transplant acceptance. In organ transplantation, CD4+ but not CD8+ T cells are essential for allo-rejection, as it has been shown that CD4+ T cells are necessary and sufficient for mediating acute rejection of heart and kidney allografts (Bolton et al., 1989Bolton E.M. Gracie J.A. Briggs J.D. Kampinga J. Bradley J.A. Cellular requirements for renal allograft rejection in the athymic nude rat.J. Exp. Med. 1989; 169: 1931-1946Crossref PubMed Scopus (66) Google Scholar, Krieger et al., 1996Krieger N.R. Yin D.P. Fathman C.G. CD4+ but not CD8+ cells are essential for allorejection.J. Exp. Med. 1996; 184: 2013-2018Crossref PubMed Scopus (272) Google Scholar). Among CD4+ T cell subsets, alloreactive T helper 1 (Th1) cells have been shown to cause allograft damage directly through Fas-Fas ligand-mediated cytotoxicity, or indirectly through inducing delayed type hypersensitivity by macrophages and promoting the activity of cytotoxic CD8+ T cells (Liu et al., 2013Liu Z. Fan H. Jiang S. CD4(+) T-cell subsets in transplantation.Immunol. Rev. 2013; 252: 183-191Crossref PubMed Scopus (91) Google Scholar). Th17 cells also mediate allograft rejection, which has been demonstrated in recipient mice lacking T-bet, the master regulator of Th1 cell differentiation (Yuan et al., 2008Yuan X. Paez-Cortez J. Schmitt-Knosalla I. D’Addio F. Mfarrej B. Donnarumma M. Habicht A. Clarkson M.R. Iacomini J. Glimcher L.H. et al.A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy.J. Exp. Med. 2008; 205: 3133-3144Crossref PubMed Scopus (256) Google Scholar). T follicular helper (Tfh) cells contribute to allograft rejection by promoting alloantibody responses (Conlon et al., 2012Conlon T.M. Saeb-Parsy K. Cole J.L. Motallebzadeh R. Qureshi M.S. Rehakova S. Negus M.C. Callaghan C.J. Bolton E.M. Bradley J.A. Pettigrew G.J. Germinal center alloantibody responses are mediated exclusively by indirect-pathway CD4 T follicular helper cells.J. Immunol. 2012; 188: 2643-2652Crossref PubMed Scopus (83) Google Scholar). By contrast, CD4+Foxp3+ regulatory T (Treg) cells protect the transplanted organs from rejection in many experimental models (Miyahara et al., 2012Miyahara Y. Khattar M. Schroder P.M. Mierzejewska B. Deng R. Han R. Hancock W.W. Chen W. Stepkowski S.M. Anti-TCRβ mAb induces long-term allograft survival by reducing antigen-reactive T cells and sparing regulatory T cells.Am. J. Transplant. 2012; 12: 1409-1418Crossref PubMed Scopus (14) Google Scholar, Safinia et al., 2015Safinia N. Scotta C. Vaikunthanathan T. Lechler R.I. Lombardi G. Regulatory T cells: serious contenders in the promise for immunological tolerance in transplantation.Front. Immunol. 2015; 6: 438Crossref PubMed Scopus (91) Google Scholar). T cell dysfunction, such as exhaustion and anergy, represents distinct T cell differentiation states following antigen encounter (Schietinger and Greenberg, 2014Schietinger A. Greenberg P.D. Tolerance and exhaustion: defining mechanisms of T cell dysfunction.Trends Immunol. 2014; 35: 51-60Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The dysfunctional differentiation of T cells involves the transcriptional induction of essential negative regulators that inhibit T cell function (Fathman and Lineberry, 2007Fathman C.G. Lineberry N.B. Molecular mechanisms of CD4+ T-cell anergy.Nat. Rev. Immunol. 2007; 7: 599-609Crossref PubMed Scopus (135) Google Scholar, Wherry and Kurachi, 2015Wherry E.J. Kurachi M. Molecular and cellular insights into T cell exhaustion.Nat. Rev. Immunol. 2015; 15: 486-499Crossref PubMed Scopus (2271) Google Scholar). For instance, dysfunctional T cells that arise during certain chronic infections and cancers sustainably express various inhibitory receptors, including programmed cell death protein 1 (PD-1), CD160, lymphocyte-activation gene 3 (LAG3), B and T lymphocyte attenuator (BTLA), and cytotoxic T lymphocyte antigen 4 (CTLA-4) (Crawford et al., 2014Crawford A. Angelosanto J.M. Kao C. Doering T.A. Odorizzi P.M. Barnett B.E. Wherry E.J. Molecular and transcriptional basis of CD4+ T cell dysfunction during chronic infection.Immunity. 2014; 40: 289-302Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, Schietinger et al., 2016Schietinger A. Philip M. Krisnawan V.E. Chiu E.Y. Delrow J.J. Basom R.S. Lauer P. Brockstedt D.G. Knoblaugh S.E. Hämmerling G.J. et al.Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis.Immunity. 2016; 45: 389-401Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). These receptors exert inhibitory effects on T cell function, and blockade of PD-1, programmed death-ligand 1 (PD-L1), or CTLA-4 has been successfully used to treat several cancer types by reversing T cell dysfunction (Zarour, 2016Zarour H.M. Reversing T-cell dysfunction and exhaustion in cancer.Clin. Cancer Res. 2016; 22: 1856-1864Crossref PubMed Scopus (258) Google Scholar). Transcription factors T-bet, Blimp-1, NFAT, and FOXO1 regulate PD-1 expression and have been implicated in T cell exhaustion and dysfunction (Wherry and Kurachi, 2015Wherry E.J. Kurachi M. Molecular and cellular insights into T cell exhaustion.Nat. Rev. Immunol. 2015; 15: 486-499Crossref PubMed Scopus (2271) Google Scholar). Interferon regulatory factor 4 (IRF4) is a member of the IRF family of transcription factors and is preferentially expressed in hematopoietic cells. It plays essential roles in many aspects of T cell, B cell, and dendritic cell differentiation and function (Huber and Lohoff, 2014Huber M. Lohoff M. IRF4 at the crossroads of effector T-cell fate decision.Eur. J. Immunol. 2014; 44: 1886-1895Crossref PubMed Scopus (141) Google Scholar, Ochiai et al., 2013Ochiai K. Maienschein-Cline M. Simonetti G. Chen J. Rosenthal R. Brink R. Chong A.S. Klein U. Dinner A.R. Singh H. Sciammas R. Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4.Immunity. 2013; 38: 918-929Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, Vander Lugt et al., 2014Vander Lugt B. Khan A.A. Hackney J.A. Agrawal S. Lesch J. Zhou M. Lee W.P. Park S. Xu M. DeVoss J. et al.Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation.Nat. Immunol. 2014; 15: 161-167Crossref PubMed Scopus (188) Google Scholar). In T cells, IRF4 is promptly expressed within hours following TCR stimulation, and its expression level is TCR affinity dependent (Man et al., 2013Man K. Miasari M. Shi W. Xin A. Henstridge D.C. Preston S. Pellegrini M. Belz G.T. Smyth G.K. Febbraio M.A. et al.The transcription factor IRF4 is essential for TCR affinity-mediated metabolic programming and clonal expansion of T cells.Nat. Immunol. 2013; 14: 1155-1165Crossref PubMed Scopus (266) Google Scholar). IRF4 controls the differentiation of Th2, Th9, Th17, Tfh, Treg, and cytotoxic effector CD8+ T cells (Bollig et al., 2012Bollig N. Brüstle A. Kellner K. Ackermann W. Abass E. Raifer H. Camara B. Brendel C. Giel G. Bothur E. et al.Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation.Proc. Natl. Acad. Sci. USA. 2012; 109: 8664-8669Crossref PubMed Scopus (138) Google Scholar, Brüstle et al., 2007Brüstle A. Heink S. Huber M. Rosenplänter C. Stadelmann C. Yu P. Arpaia E. Mak T.W. Kamradt T. Lohoff M. The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4.Nat. Immunol. 2007; 8: 958-966Crossref PubMed Scopus (547) Google Scholar, Cretney et al., 2011Cretney E. Xin A. Shi W. Minnich M. Masson F. Miasari M. Belz G.T. Smyth G.K. Busslinger M. Nutt S.L. Kallies A. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells.Nat. Immunol. 2011; 12: 304-311Crossref PubMed Scopus (438) Google Scholar, Huber et al., 2008Huber M. Brüstle A. Reinhard K. Guralnik A. Walter G. Mahiny A. von Löw E. Lohoff M. IRF4 is essential for IL-21-mediated induction, amplification, and stabilization of the Th17 phenotype.Proc. Natl. Acad. Sci. USA. 2008; 105: 20846-20851Crossref PubMed Scopus (221) Google Scholar, Staudt et al., 2010Staudt V. Bothur E. Klein M. Lingnau K. Reuter S. Grebe N. Gerlitzki B. Hoffmann M. Ulges A. Taube C. et al.Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.Immunity. 2010; 33: 192-202Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, Yao et al., 2013Yao S. Buzo B.F. Pham D. Jiang L. Taparowsky E.J. Kaplan M.H. Sun J. Interferon regulatory factor 4 sustains CD8(+) T cell expansion and effector differentiation.Immunity. 2013; 39: 833-845Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, Zheng et al., 2009Zheng Y. Chaudhry A. Kas A. deRoos P. Kim J.M. Chu T.T. Corcoran L. Treuting P. Klein U. Rudensky A.Y. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses.Nature. 2009; 458: 351-356Crossref PubMed Scopus (720) Google Scholar). Irf4-deficient T cells exhibit a severe functional defect in T cell-mediated responses, including microbial infection, allergy, graft-versus-host reaction, and autoimmunity (Brüstle et al., 2007Brüstle A. Heink S. Huber M. Rosenplänter C. Stadelmann C. Yu P. Arpaia E. Mak T.W. Kamradt T. Lohoff M. The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4.Nat. Immunol. 2007; 8: 958-966Crossref PubMed Scopus (547) Google Scholar, Grusdat et al., 2014Grusdat M. McIlwain D.R. Xu H.C. Pozdeev V.I. Knievel J. Crome S.Q. Robert-Tissot C. Dress R.J. Pandyra A.A. Speiser D.E. et al.IRF4 and BATF are critical for CD8+ T-cell function following infection with LCMV.Cell Death Differ. 2014; 21: 1050-1060Crossref PubMed Scopus (50) Google Scholar, Huber and Lohoff, 2014Huber M. Lohoff M. IRF4 at the crossroads of effector T-cell fate decision.Eur. J. Immunol. 2014; 44: 1886-1895Crossref PubMed Scopus (141) Google Scholar, Mittrücker et al., 1997Mittrücker H.W. Matsuyama T. Grossman A. Kündig T.M. Potter J. Shahinian A. Wakeham A. Patterson B. Ohashi P.S. Mak T.W. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function.Science. 1997; 275: 540-543Crossref PubMed Scopus (5) Google Scholar, Staudt et al., 2010Staudt V. Bothur E. Klein M. Lingnau K. Reuter S. Grebe N. Gerlitzki B. Hoffmann M. Ulges A. Taube C. et al.Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.Immunity. 2010; 33: 192-202Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Here we examined the role of IRF4 in transplant rejection and acceptance. We found that all fully MHC-mismatched heart allografts survived indefinitely in Irf4-deficient mice. This stable engraftment in Irf4-deficient recipients involved the progressive establishment of effector CD4+ T cell dysfunction. Mechanistically, IRF4 repressed a group of previously defined molecules associated with CD4+ T cell dysfunction, including PD-1 and Helios. In particular, in the absence of IRF4, chromatin accessibility and binding of Helios at PD-1 cis-regulatory elements were increased, resulting in enhanced PD-1 expression and CD4+ T cell dysfunction. Although the dysfunctional state of Irf4-deficient T cells was initially reversible by blockade of PD-1-PD-L1 pathway, it progressively evolved into a “terminal” irreversible state within 30 days post-transplant. Taken together, our results revealed that IRF4 repressed the dysfunctional differentiation of CD4+ T cells. Induction of T cell dysfunction by targeting IRF4 during transplantation may be a therapeutically relevant strategy for achieving transplant acceptance. IRF4 is a key transcription factor for translating TCR signaling into proper T cell responses (Huber and Lohoff, 2014Huber M. Lohoff M. IRF4 at the crossroads of effector T-cell fate decision.Eur. J. Immunol. 2014; 44: 1886-1895Crossref PubMed Scopus (141) Google Scholar), but its role in T cell-mediated transplant rejection remains unclear. Here we first assessed IRF4 expression in T cells in response to heart transplantation. BALB/c hearts were transplanted into fully MHC-mismatched C57BL/6 (B6) recipients. Splenocytes and graft-infiltrating cells were harvested and analyzed at 7 days post-transplant when heart allografts were rejected. We found that graft-infiltrating T cells expressed a significantly high mean fluorescence intensity (MFI) of IRF4, while splenic T cells from transplanted mice had a moderate increase in IRF4 expression compared to that of naive B6 mice (Figures 1A and 1B ). The majority of graft-infiltrating T cells had lost CD62L expression but expressed T cell activation markers CD44, glucose transporter 1 (GLUT1), and CD98 (Figures S1A and S1B). Thus, IRF4 was highly expressed in graft-infiltrating T cells and was correlated to the activation status of these cells. To investigate whether IRF4 expression in T cells plays a role in transplant rejection, we transplanted BALB/c hearts into T cell-specific IRF4 knockout (Irf4fl/flCd4-Cre; B6 background) or wild-type (WT) B6 mice. None of the Irf4fl/flCd4-Cre mice rejected their BALB/c heart allografts (median survival time [MST] of > 100 days; n = 6), whereas WT B6 mice rejected BALB/c hearts acutely (MST = 7.17 ± 0.41 days; n = 6) (Figure 1C). Histology of heart allografts harvested from Irf4fl/flCd4-Cre recipient mice at days 7 and 100 post-transplant showed intact myocytes with minimal cellular infiltration and vasculopathy (Figure 1D). Hence, selective ablation of IRF4 in T cells abrogated their ability to reject heart allografts, which provides a potential prospect for achieving graft acceptance. To determine whether a lack of functional T cells in Irf4fl/flCd4-Cre mice accounts for the graft acceptance, Irf4fl/flCd4-Cre mice were adoptively transferred with 2 million WT B6 CD4+ or CD8+ T cells or with 20 million Irf4‒/‒ CD4+ or CD8+ T cells 1 day prior to BALB/c heart transplantations. Irf4fl/flCd4-Cre recipients transferred with 2 million WT B6 CD4+ T cells acutely rejected their BALB/c heart allografts (MST = 7.83 ± 0.41 days), whereas none of the Irf4fl/flCd4-Cre recipients in other groups rejected the heart allografts (Figure 2A). These results indicated that in our model the lack of functional CD4+ (but not CD8+) T cells was essential for heart allograft acceptance and that increasing the number of dysfunctional Irf4-deficient CD4+ T cells failed to restore transplant rejection. CD4+CD25+Foxp3+ Treg cells are essential to maintain long-term allograft survival in many experimental models (Miyahara et al., 2012Miyahara Y. Khattar M. Schroder P.M. Mierzejewska B. Deng R. Han R. Hancock W.W. Chen W. Stepkowski S.M. Anti-TCRβ mAb induces long-term allograft survival by reducing antigen-reactive T cells and sparing regulatory T cells.Am. J. Transplant. 2012; 12: 1409-1418Crossref PubMed Scopus (14) Google Scholar). To determine whether Treg cells contribute to the allograft acceptance in Irf4fl/flCd4-Cre mice, we transplanted BALB/c hearts into Irf4fl/flCd4-Cre mice and treated them with the PC61 anti-CD25 mAb either on days −1, 3, and 6 (induction phase of graft acceptance) or on days 50, 53, and 56 (maintenance phase) or with a control IgG on days −1, 3, and 6 post-transplant. Injection of PC61 mAb eliminated approximately 70% of CD4+FoxP3+ cells in peripheral blood of recipient mice 1 day after treatment completed (data not shown). Nevertheless, this partial Treg cell depletion during the induction or maintenance phase did not abrogate permanent allograft survival in Irf4fl/flCd4-Cre mice, which was the same as that in control IgG group (MST of > 100 days; n = 5 each group) (Figure 2B). We then focused on identifying intrinsic changes of Irf4-deficient T cells responsible for long-term allograft acceptance. BALB/c hearts were transplanted into Irf4fl/flCd4-Cre or WT B6 mice. Before transplantation, Irf4fl/flCd4-Cre mice had significantly more T cells in spleens than did WT B6 mice. At day 7 post-transplant, T cell numbers in the spleens of Irf4fl/flCd4-Cre mice remained largely unchanged (similar to that in un-transplanted Irf4fl/flCd4-Cre mice), while the number of splenic T cells (particularly CD8+ T cells) in WT recipients was increased (Figure S1C). These results indicated that the expansion of alloreactive T cells in Irf4fl/flCd4-Cre recipients was impaired. Moreover, frequencies of CD4+CD62LloCD44+, CD8+CD62LloCD44+, and CD4+Foxp3+ splenocytes from Irf4fl/flCd4-Cre recipients were significantly lower than those of WT recipients (Figure S1C). CD4+BCL6+CXCR5+ Tfh cells, CD19loCD138+ plasma cells, and CD19+GL7+PNA+ germinal center B cells were absent in the spleens of Irf4fl/flCd4-Cre recipients but were clearly detected in WT recipients at day 9 post-transplant (Figure S1D). Hence, IRF4 was essential for the induction of Tfh cell response to heart transplant. An adoptive co-transfer model was used to further assess the intrinsic changes of Irf4-deficient T cells. CD45.1+ WT T cells and CD45.2+ Irf4‒/‒ T cells were co-injected at a 1:1 ratio into B6.Rag1‒/‒ mice 1 day before receiving BALB/c heart allografts (Figure 2C). The heart grafts were rejected between 9 and 15 days after transplant. At day 9 post-transplant, about 30% transferred cells in spleens were Irf4‒/‒ T cells (Figure 2D). Virtually all graft-infiltrating T cells were CD45.1+ WT T cells (Figure 2E), demonstrating that Irf4‒/‒ T cells lost the ability to infiltrate allografts. The infiltrating Irf4‒/‒ T cells were too few to be compared with WT T cells, and so we compared the co-injected T cells in the spleens. Both CD4+ and CD8+ Irf4‒/‒ T cells did not downregulate CD62L and barely expressed an effector marker KLRG1. CD4+ Irf4‒/‒ T cells also failed to upregulate CD44 expression (Figure 2F). In addition, the frequencies of IFN-γ- and IL-17-producing cells within CD4+ Irf4‒/‒ T cells were significantly lower than those of CD4+ WT T cells (Figure 2G), and the frequency of IFN-γ-producing cells within CD8+ Irf4‒/‒ T cells was also lower than that of CD8+ WT T cells (Figure 2H). Thus, IRF4 deficiency inhibited the expression of effector T cell markers and the production of signature cytokines for effector T cells in response to heart transplantation. The frequency of Foxp3+ cells within CD4+ Irf4‒/‒ splenic T cells was lower than that of CD4+ WT splenocytes (Figure 2G). Next, we repeated these experiments by separately transferring sorted CD4+ WT, CD4+ Irf4‒/‒, CD8+ WT, or CD8+ Irf4‒/‒ T cells into B6.Rag1‒/‒ mice 1 day prior to BALB/c heart transplantation (Figure S2A). Ex vivo analysis of transferred cells was performed on day 9 after transplant. Consistent with results from the co-transfer model, separately injected CD4+ and CD8+ Irf4‒/‒ T cells also lost their ability to infiltrate allografts (Figure S2B). Compared to the separately transferred WT T cells in spleens, CD4+ and CD8+ Irf4‒/‒ T cells largely maintained CD62L expression, barely expressed KLRG1, and produced significantly less IFN-γ (Figures S2C–S2F). CD4+ Irf4‒/‒ T cells also produced significantly less IL-17 and expressed less Foxp3 (Figure S2E). Taken together, IRF4 promoted effector T cell differentiation and infiltration into heart allografts. We showed above that the lack of functional CD4+ T cells accounted for the graft acceptance in Irf4fl/flCd4-Cre mice, and therefore we focus on defining the intrinsic mechanism underlying the dysfunction of Irf4-deficient CD4+ T cells. We measured expression of inhibitory and costimulatory receptors on Irf4‒/‒ or WT CD4+ T cells 1 day after in vitro activation. Compared to WT CD4+ T cells, Irf4‒/‒ CD4+ T cells expressed higher MFIs of exhaustion and anergy signatures including PD-1, CD160, CD73, and folate receptor 4 (FR4) (Martinez et al., 2012Martinez R.J. Zhang N. Thomas S.R. Nandiwada S.L. Jenkins M.K. Binstadt B.A. Mueller D.L. Arthritogenic self-reactive CD4+ T cells acquire an FR4hiCD73hi anergic state in the presence of Foxp3+ regulatory T cells.J. Immunol. 2012; 188: 170-181Crossref PubMed Scopus (65) Google Scholar, Wherry and Kurachi, 2015Wherry E.J. Kurachi M. Molecular and cellular insights into T cell exhaustion.Nat. Rev. Immunol. 2015; 15: 486-499Crossref PubMed Scopus (2271) Google Scholar) and also expressed similar or slightly lower MFIs of BTLA and CTLA-4. Profoundly, another essential exhaustion marker, LAG-3, was significantly decreased on Irf4‒/‒ CD4+ T cells (Figure 3A). We used microarray analysis to compare the gene expression profiles between Irf4‒/‒ and WT CD4+ T cells following 2 day in vitro activation. Among 672 differentially expressed genes, 438 were increased in activated Irf4‒/‒ T cells and were significantly enriched in the Gene Ontology (GO) categories of “negative regulation of biological process” and “negative regulation of cell activation” (Figure 3C). Ikzf2 (encoding Helios), Pdcd1 (encoding PD-1), and Cd160 were among the highest upregulated genes in activated Irf4‒/‒ CD4+ T cells when compared to activated WT CD4+ T cells (Figure 3B), which were confirmed by quantitative real-time PCR (Figure 3D). Helios is a signature protein for T cell dysfunction (Crawford et al., 2014Crawford A. Angelosanto J.M. Kao C. Doering T.A. Odorizzi P.M. Barnett B.E. Wherry E.J. Molecular and transcriptional basis of CD4+ T cell dysfunction during chronic infection.Immunity. 2014; 40: 289-302Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, Singer et al., 2016Singer M. Wang C. Cong L. Marjanovic N.D. Kowalczyk M.S. Zhang H. Nyman J. Sakuishi K. Kurtulus S. Gennert D. et al.A distinct gene module for dysfunction uncoupled from activation in tumor-infiltrating T cells.Cell. 2016; 166: 1500-1511.e9Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). We found that Helios protein was absent in activated WT CD4+ T cells but was expressed in more than 50% activated Irf4‒/‒ CD4+ T cells (Figure 3E). Collectively, IRF4 repressed a group of previously defined molecules associated with CD4+ T cell dysfunction. Given that Pdcd1 was among the highest upregulated genes in Irf4‒/‒ versus WT CD4+ T cells after activation, we aimed to understand the regulation of PD-1 expression by IRF4. We observed that PD-1 expression on Irf4‒/‒ CD4+ T cells was progressively increased from day 0 to day 3 upon in vitro activation (Figure 4A) and was much higher than that of co-cultured CD45.1+ WT CD4+ T cells (Figure 4B). To further examine the role of IRF4 in PD-1 expression, activated Irf4‒/‒ CD4+ T cells were transduced with a retroviral vector expressing IRF4-green fluorescent protein (GFP) or a control vector expressing just GFP. As shown in Figure 4C, transduction of IRF4 into activated Irf4‒/‒ CD4+ T cells (detected by GFP expression) led to a marked inhibition of PD-1 expression when compared with GFP control transduction. Thus, IRF4 expression in activated T cells mediated repression of PD-1. To determine how IRF4 represses PD-1 expression, we performed chromatin immunoprecipitation (ChIP) analysis. In activated WT CD4+ T cells (expressing IRF4), we did not detect specific enrichment of IRF4 at the putative binding sites upstream of Pdcd1 or at a set of known cis-elements of Pdcd1, including two upstream conserved regions (CR-B and CR-C) as well as two regions located ‒3.7 and +17.1 kb from the Pdcd1 transcription start site (Figure S3; Bally et al., 2016Bally A.P. Austin J.W. Boss J.M. Genetic and epigenetic regulation of PD-1 expression.J. Immunol. 2016; 196: 2431-2437Crossref PubMed Scopus (131) Google Scholar). These data suggested that the repression effect of IRF4 on PD-1 expression was unlikely to be related to its transcriptional activity. We next investigated whether histone modifications are involved in the regulation of PD-1 expression by IRF4. As shown in Figure 4D, H3 acetylation (H3Ac) was significantly increased at the −3.7 site and the CR-B and CR-C regions, whereas H4 acetylation (H4Ac) and H3 lysine 4 trimethylation (H3K4me3) were markedly increased at the −3.7 site and the CR-C region in activated Irf4‒/‒ CD4+ T cells compared with those in activated WT CD4+ T cells. H3Ac, H4Ac, and H3K4me3 are all active histone marks. The expression of a repressive histone mark, H3 lysine 9 trimethylation (H3K9me3), displayed no changes at the cis-elements of Pdcd1 in Irf4‒/‒ relative to WT CD4+ T cells. Therefore, IRF4 deficiency in activated CD4+ T cells induced an active chromatin state at the critical cis-elements of Pdcd1, correlating to the elevated PD-1 expression. In activated Irf4‒/‒ CD4+ T cells, most PD-1hi cells were Helios positive (Figure 4E). We therefore investigated whether Helios regulates PD-1 expression. This possibility was supported by a ChIP assay, which detected the binding of Helios to the cis-elements of Pdcd1 in activated Irf4‒/‒ CD4+ T cells (Figure 4F). Furthermore, activated WT CD4+ T cells (with minimal Helios expression) were transduced with a retroviral vector expressing Helios-GFP or just GFP. We found that transduction of Helios into activated WT CD4+ T cells resulted in a marked increase of PD-1 expression when compared with GFP control transduction (Figure 4G). Activated Irf4‒/‒ CD4+ T cells (with Helios expression) were also transduced with a retroviral vector co-expressing GFP and short hairpin RNA (shRNA) sequences for Helios (sh-Helios) or expressing GFP alone (sh-Ctrl). As indicated by GFP+ cells, transduction with sh-Helios decreased PD-1 expression in Irf4‒/‒ CD4+ T cells (Figure 4H). Thus, Helios promoted PD-1 expression. Taken together, IRF4 deletion progressively increased PD-1 expression on activated T cells, which was associated with the increased chromatin accessibility and Helios binding to PD-1 cis-regulatory elements. TCR-transgenic TEa CD4+ T cells (B6 background) recognize a BALB/c I-Eα allopeptide presented by B6 APCs; mice containing only TEa T cells were able to reject BALB/c skin allografts (Gupta et al., 2011Gupta S. Balasubramanian S. Thornley T.B. Strom T.B. Kenny J.J. Direct pathway T-cell alloactivation is more rapid than indirect pathway alloactivation.Transplantation. 2011; 91: e65-e67Crossref PubMed Scopus (5) Google Scholar). We found that adoptive transfer of WT TEa but not Irf4‒/‒ TEa cells induced rejection of BALB/c hearts in Irf4fl/flCd4-Cre recipients (Figure 5A). To determine whether immune checkpoint PD-1 contributes to the dysfunction of alloantigen-specific Irf4-deficient CD4+ T cells in transplantation, we assessed PD-1 expression on Irf4‒/‒ TEa versus WT TEa cells by transferring and tracking these cells in CD45.1+ B6 congenic mice. As shown in Figure 5B, CD45.1+ B6 mice were transferred with either CD45.2+ WT TEa or CD45.2+ Irf4‒/‒ TEa cells on day −1 and were transplanted with BALB/c hearts or left un-transplanted on day 0. Splenocytes were analyzed on day 6 (Figure 5B). In CD45.1+ B6 mice without BALB/c heart grafting, TEa CD4+ T cells neither proliferated nor expressed PD-1. In heart transplanted mice, Irf4‒/‒ TEa cells exhibited higher PD-1 expression and a lower proliferation rate than did WT TEa cells (Figure 5B). Thus, IRF4 deficiency promoted PD-1 expression on alloantigen-specific T cells, which was associated with decreased cell proliferation. Intracellular CTLA-4 expression was also detectable in WT and Irf4‒/‒ TEa cells in transplanted groups, and WT TEa" @default.
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• W2773141414 date "2017-12-01" @default.
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• W2773141414 title "Ablation of Transcription Factor IRF4 Promotes Transplant Acceptance by Driving Allogenic CD4+ T Cell Dysfunction" @default.
• W2773141414 cites W1547956541 @default.
• W2773141414 cites W1582225030 @default.
• W2773141414 cites W1652539448 @default.
• W2773141414 cites W1814637494 @default.
• W2773141414 cites W1855786281 @default.
• W2773141414 cites W1910012553 @default.
• W2773141414 cites W1964081728 @default.
• W2773141414 cites W1966818078 @default.
• W2773141414 cites W1973563476 @default.
• W2773141414 cites W2001042507 @default.
• W2773141414 cites W2001105504 @default.
• W2773141414 cites W2009675211 @default.
• W2773141414 cites W2011873300 @default.
• W2773141414 cites W2017104717 @default.
• W2773141414 cites W2022687427 @default.
• W2773141414 cites W2023003954 @default.
• W2773141414 cites W2043776386 @default.
• W2773141414 cites W2045067923 @default.
• W2773141414 cites W2045348005 @default.
• W2773141414 cites W2045468537 @default.
• W2773141414 cites W2046781420 @default.
• W2773141414 cites W2047604395 @default.
• W2773141414 cites W2049886038 @default.
• W2773141414 cites W2050317788 @default.
• W2773141414 cites W2058950182 @default.
• W2773141414 cites W2074813958 @default.
• W2773141414 cites W2080935386 @default.
• W2773141414 cites W2083380291 @default.
• W2773141414 cites W2088275003 @default.
• W2773141414 cites W2091954081 @default.
• W2773141414 cites W2125164691 @default.
• W2773141414 cites W2128758225 @default.
• W2773141414 cites W2140748367 @default.
• W2773141414 cites W2142335261 @default.
• W2773141414 cites W2145026575 @default.
• W2773141414 cites W2146488955 @default.
• W2773141414 cites W2156177776 @default.
• W2773141414 cites W2164526800 @default.
• W2773141414 cites W2166662937 @default.
• W2773141414 cites W2191493147 @default.
• W2773141414 cites W2223436478 @default.
• W2773141414 cites W2279561773 @default.
• W2773141414 cites W2303235652 @default.
• W2773141414 cites W2336076245 @default.
• W2773141414 cites W2336635113 @default.
• W2773141414 cites W2436440352 @default.
• W2773141414 cites W2510606169 @default.
• W2773141414 cites W2514996992 @default.
• W2773141414 cites W2534725153 @default.
• W2773141414 cites W2572174216 @default.
• W2773141414 cites W2582906781 @default.
• W2773141414 cites W4301240852 @default.
• W2773141414 doi "https://doi.org/10.1016/j.immuni.2017.11.003" @default.
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