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The Importance of Cooperation: Partnerless NFAT Induces T Cell Exhaustion Bertram Bengsch1 and E. John Wherry1,* 1Institute for Immunology, Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2015.01.023

Downstream of costimulatory pathways, complexes of transcription factors NFAT and AP-1 promote effector T cell differentiation. In this issue of Immunity, Martinez et al. report that monomeric NFAT binding in the absence of a transcriptional partner induces a gene expression program involved in T cell exhaustion. T cell exhaustion is a type of T cell dysfunction that occurs in many chronic infections and cancer. Hallmarks of T cell exhaustion include reduced effector function, expression of inhibitory receptors (e.g., programmed cell death-1 [PD-1]), and distinct transcriptional and metabolic states. Recently, checkpoint blockade targeting inhibitory receptors expressed by exhausted T (Tex) cells such as PD-1 has shown great promise in patients with advanced cancer. In contrast to effector T (Tef) cells generated in the presence of adequate T cell receptor (TCR) stimulation and costimulation, Tex cells arise in the context of high and prolonged antigen stimulation and excess of coinhibitory over costimulatory signals. However, our understanding of how T cells integrate this information into the transcription of ‘‘exhaustion’’ versus ‘‘effector’’ gene programs remains limited. Several transcriptional circuits have been implicated in Tex cells including transcription factors T-bet and Eomes, Blimp1, hypoxia-inducible factors, Foxo1, the AP-1 family member BATF, and NFAT (Kahan et al., 2015). Some transcription

factors like BATF might regulate early TCR-dependent expression of other transcription factors shared between Tex and Tef cells. Other transcriptional circuits have been described in Tex versus functional T cells. Transcriptional network analysis highlighted the context-dependent nature of key transcription factors expressed by Tex cells, indicating that these transcription factors can be linked to differential gene expression in Tex compared to functional T cells (Doering et al., 2012). For example, quantitative changes in T-box transcription factors (e.g., T-bet, Eomes) are associated with exhaustion compared to memory development. Despite this work it remains unclear what the earliest transcriptional control points are that govern the development of functional versus dysfunctional T cells. In this issue of Immunity, Martinez et al. (2015) show that different modes of transcription factor NFAT binding underlie the expression of exhausted versus effector transcriptional programs (Figure 1). Lymphocytes express NFAT family members 1, 2, and 4 that get activated by TCRinduced, calcium-dependent, calcineurin-

mediated dephosphorylation of cytosolic NFAT and subsequent nuclear translocation. In addition to a DNA binding domain, NFATs contain domains that allow protein-protein interactions with other transcriptional cofactors. Such a system allows integration of transcriptional inputs downstream of costimulatory and other regulatory signals. For example, in Tef cells, binding of AP-1 complexes formed downstream of costimulatory signaling pathways is critical for transcription of T cell effector proteins, such as interleukin-2 (IL-2) and interferon-g (IFN-g). Importantly, lack of transcriptional cooperation between NFAT and other transcription factors is involved in T cell anergy, a distinct type of dysfunctional T cells that contributes to peripheral tolerance (Mu¨ller and Rao, 2010). Anergic CD4+ T cells are induced by TCR engagement without costimulation, a scenario that activates NFAT in the absence of AP-1 and initiates transcription of anergyrelated genes. A different regulatory mechanism involving NFAT occurs in regulatory T cells, where NFAT co-binding with FOXP3 results in transcriptional

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Previews Cosmulatory receptor (e.g. CD28)

+ TCR

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Effector genes (encoding IFN-γ, IL-2)

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TGGAAAnnnTGAG/CTCA Composite binding mof Monomeric binding mof

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+ Figure 1. Cooperative NFAT:AP-1 Transcription Factor Binding Sites Are Preferentially Found at Effector Gene Transcriptional Start Sites In the context of TCR signaling and appropriate costimulation (e.g., via CD28), NFAT and AP-1 will cooperate to induce an effector gene program. However, in T cell exhaustion, lack of functional AP-1 availability will result in NFAT binding to monomeric binding sites located at TSSs of exhaustion-related genes, such as multiple inhibitory receptors and immunoregulatory genes.

repression, rather than induction, of IL-2 (Mu¨ller and Rao, 2010). But do exhausted cells actually require NFAT? Some previous evidence suggested a role for NFAT in Tex cells (Kahan et al., 2015), yet exactly how this transcription factor functioned in this context was unclear. In this issue of Immunity, Martinez et al. (2015) demonstrate markedly diminished expression of inhibitory receptors and effector cytokines in activated T cells genetically deficient in NFAT1 and 2. The residual expression of these proteins was further reduced by NFAT4 knockdown, suggesting that all calcium-regulated lymphocytic NFAT family members contribute to both the effector and exhaustion program. Even during LCMV clone 13 infection, a widely used mouse model of chronic infection

and T cell exhaustion, exhaustion did not develop without functional NFAT1 and 2, though whether NFAT-deficient T cells undergo normal initial activation and expansion during infection is not clear. Nevertheless, these findings demonstrate an NFAT requirement for the development of T cell exhaustion. The authors employed an elegant approach to study the properties of NFAT binding to promoter regions in the absence of AP-1 by using a constitutively active variant of NFAT1 mutated to abolish AP-1 binding (CA-RIT-NFAT1). This construct is expected to bind in a monomeric fashion (i.e., without AP-1) to DNA. In-vitro-activated T cells expressing this construct displayed reduced IL-2 production and attenuated T cell signaling. Further, expression of CA-RIT-NFAT1

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interfered with T cell effector function in two in vivo models. First, CA-RIT-NFAT1expressing CD8+ T cells were impaired in their ability to protect during Listeria monocytogenes infection. Similarly to Tex cells described in chronic viral infections, CA-RIT-NFAT1-transduced cells had higher expression of inhibitory receptors including PD-1, Tim-3, and LAG-3. Second, CA-RIT-NFAT-1-transduced T cells were unable to reject CT26 tumor cells and again displayed high inhibitory receptor coexpression. These findings indicate that NFAT1 binding in the absence of AP-1 fosters a program of T cell dysfunction resembling exhaustion. To define the molecular and transcriptional mechanisms by which NFAT binding in the absence of AP-1 regulated exhaustion, the authors performed genome-wide RNA-seq of CA-RIT-NFAT1-transduced cells. Large alterations of the transcriptome were identified compared to control transduced cells, affecting 2,000 genes that overlapped significantly with exhausted and anergic gene sets. Importantly, several key molecules involved in T cell exhaustion were directly upregulated by CA-RIT-NFAT1, including inhibitory receptors (e.g., LAG-3, Tim-3, PD-1), transcription factors (e.g., Blimp-1), and other immunoregulatory proteins (e.g., diacylglycerol kinase). These data suggest that NFAT binding in the absence of AP-1 drives expression of a key set of exhaustion-related genes. Additional ChIP-seq studies demonstrated that CA-RIT-NFAT1 bound to transcriptional start sites (TSSs) of genes identified as upregulated by RNA-seq, such as Lag3. No such association was found with downregulated genes, suggesting that monomeric NFAT1 functions as a positive regulator of gene expression. In addition, WT NFAT1 bound to approximately twice as many genomic sites as CA-RIT-NFAT1, indicating that at least half the genes regulated by WT NFAT probably require additional binding of transcriptional cofactors, such as AP-1. Finally, a composite NFAT1:AP-1 binding element was enriched in regions bound by WT NFAT1 but not CA-RIT-NFAT1. In contrast, monomeric NFAT1 binding elements were dominant in the setting of CA-RIT-NFAT1 binding, including binding to TSSs of inhibitory receptor genes (e.g., PD-1, CTLA-4, Tim-3, LAG-3). Thus,

Immunity

Previews NFAT-regulated genes implicated in effector function are typically characterized by promoters and/or enhancers harboring a composite NFAT1:AP-1 binding element, whereas NFAT-regulated genes involved in T cell dysfunction display monomeric NFAT1 binding motifs. These findings imply that monomeric NFAT binding motifs constitute a genetic coding for genes involved in T cell dysfunction such as exhaustion. These data suggest that T cell receptor stimulation in the presence of insufficient positive costimulation (i.e., a major source for AP-1 signals) could be important for T cell exhaustion. This notion is supported by results from the LCMV model showing that high amounts of antigen stimulation, including that presented by non-professional antigen-presenting cells, are linked to T cell exhaustion (Mueller and Ahmed, 2009). Indeed, a major role for inhibitory receptors such as PD-1 might be to disrupt the AP-1 signals generated through positive signaling cascades (Quigley et al., 2010) and to shift the relative ratio of nuclear NFAT:canonical AP-1 complexes to an excess of ‘‘partnerless’’ NFAT favoring exhaustion (Figure 1). It should be noted, however, that NFATdependent gene expression is not sufficient for the development of all aspects of T cell exhaustion, but rather seems to contribute to an early transcriptional check point. Exhaustion is a progressive process. For example, T cells primed during chronic LCMV infection maintain fate flexibility during the effector phase and can develop into functional memory T cells if removed from infection within 1 week. This plasticity wanes over time, however, suggesting that an irre-

versible program of exhaustion becomes further established during extended chronic stimulation. These observations are consistent with the role for Eomes found in exhaustion (Paley et al., 2012) that appears to be independent of the monomeric NFAT pathway described in the current work. Nevertheless, the early NFAT-driven expression of inhibitory receptors (such as PD-1) and other regulatory proteins illustrated in this new work could allow for the integration of additional feed-forward signals that reinforce development of exhaustion (e.g., after PDL1:PD-1 ligation). These signals might then add additional layers to the transcriptional program of T cell exhaustion. One example might be the control of cell fate and metabolism exerted through signals mediated by the PD-1 pathway. Indeed, a recent study described a feedback loop for PD-1 signaling that activates the transcription factor FoxO1, which in turn sustains PD-1 expression and homeostasis of exhausted T cell populations (Staron et al., 2014). A better understanding of the role of NFAT:AP-1 transcriptional interactions might have therapeutic implications. For example, this new work suggests that reestablishment of cooperative NFAT1:AP1 binding might be an effective strategy to reverse T cell exhaustion. Induction of AP-1 family members has been reported downstream of several costimulatory pathways, such as CD28 or TNF family members OX40, 4-1BB, and CD27 (Chen and Flies, 2013). Indeed, significant increases in T cell function have been reported when costimulatory pathways were activated alone or in combination

with PD-1 blockade (Vezys et al., 2011). Thus, these new findings not only shed light on the transcriptional role for NFAT in T cell exhaustion and provide a genomic and mechanistic understanding for this process, but could also provide a framework for rationally selecting optimal combinations of immunotherapy in settings of T cell exhaustion like cancer and chronic infections. REFERENCES Chen, L., and Flies, D.B. (2013). Nat. Rev. Immunol. 13, 227–242. Doering, T.A., Crawford, A., Angelosanto, J.M., Paley, M.A., Ziegler, C.G., and Wherry, E.J. (2012). Immunity 37, 1130–1144. Kahan, S.M., Wherry, E.J., and Zajac, A.J. (2015). Virology. Published online January 22, 2015. http://dx.doi.org/10.1016/j.virol.2014.12.033. Martinez, G.J., Pereira, R.M., A¨ijo¨, T., Kim, E.Y., Marangoni, F., Pipkin, M.E., Togher, S., Heissmeyer, V., Zhang, Y.C., Crotty, S., et al. (2015). Immunity 42, this issue, 265–278. Mueller, S.N., and Ahmed, R. (2009). Proc. Natl. Acad. Sci. USA 106, 8623–8628. Mu¨ller, M.R., and Rao, A. (2010). Nat. Rev. Immunol. 10, 645–656. Paley, M.A., Kroy, D.C., Odorizzi, P.M., Johnnidis, J.B., Dolfi, D.V., Barnett, B.E., Bikoff, E.K., Robertson, E.J., Lauer, G.M., Reiner, S.L., and Wherry, E.J. (2012). Science 338, 1220–1225. Quigley, M., Pereyra, F., Nilsson, B., Porichis, F., Fonseca, C., Eichbaum, Q., Julg, B., Jesneck, J.L., Brosnahan, K., Imam, S., et al. (2010). Nat. Med. 16, 1147–1151. Staron, M.M., Gray, S.M., Marshall, H.D., Parish, I.A., Chen, J.H., Perry, C.J., Cui, G., Li, M.O., and Kaech, S.M. (2014). Immunity 41, 802–814. Vezys, V., Penaloza-MacMaster, P., Barber, D.L., Ha, S.J., Konieczny, B., Freeman, G.J., Mittler, R.S., and Ahmed, R. (2011). J. Immunol. 187, 1634–1642.

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The importance of cooperation: partnerless NFAT induces T cell exhaustion.

Downstream of costimulatory pathways, complexes of transcription factors NFAT and AP-1 promote effector T cell differentiation. In this issue of Immun...
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