The Transcription Factor NFAT Promotes Exhaustion of Activated CD8 + T Cells

“Barcoded librarieswere sequenced in aSOLID4 sequencer (Applied Biosystems) and the reads were mapped by Bowtie (Langmead et al., 2009) to the mm9 genome in color-space.”

“, 2011), and in CD8 T cells in the context of prolonged low-grade antigenic stimulation and/or inflammation, as encountered in chronic viral infections and cancer (Schietinger and Greenberg, 2014; Wherry, 2011).”

“, 2006), and blocking this pathway during chronic LCMV infection reinvigorates virus-specific CD8 T cell responses, resulting in lower viral loads (Barber et al., 2006).”

“Notably, most NFAT1 ChIP-seq peaks did not overlap with CARIT-NFAT1 ChIP-seq peaks (Figure 4A, left and middle), suggesting that at these sites, NFAT binding is strongly stabilized by AP-1 (probably by 20-fold ormore) (Rao et al., 1997).”

“…in CD8+ and CD4+ T cells by CA-RIT-NFAT1 and those observed in exhausted CD8+ T cells and anergic CD4+ T cells in vivo (22/56 genes and 99/371 genes, respectively; p value < 10 10) (Figures 2E, 2F, and S3E; Tables 1, S1, and S2; Doering et al., 2012; Okamura et al., 2009; Wherry et al., 2007).”

“Moreover, antibodies to PD-1 and CTLA-4 have emerged as surprisingly effective agents for cancer immunotherapy (Schietinger and Greenberg, 2014), and individual or combined blockade of TIM3, PD-1, and LAG3 with antagonist antibodies has been shown to reverse exhaustion efficiently in CD8 T cells (Blackburn et al., 200…”

“Moreover, antibodies to PD-1 and CTLA-4 have emerged as surprisingly effective agents for cancer immunotherapy (Schietinger and Greenberg, 2014), and individual or combined blockade of TIM3, PD-1, and LAG3 with antagonist antibodies has been shown to reverse exhaustion efficiently in CD8 T cells (Blackburn et al., 200…”

“The ability to participate inmultiple transcriptional complexes allows NFAT to contribute to different transcriptional programs depending on the cell type and signaling context in which it is activated (Hogan et al., 2003).”

“Function In Vivo To test the biological effects of expressing CA-RIT-NFAT1 in CD8+ T cells, we utilized an in vivo Listeria protection assay (modified from Kaech et al., 2003) (Figures 1A and S1I).”

“The combined action of these negative regulators is likely to underlie the diminished activation of ZAP-70 and PLCg1 and the consequent decrease in Ca2+ influx that we observe in CARIT-NFAT1-expressing T cells.”

“The transcription factor nuclear factor of activatedT cells (NFAT) is well establishedasakey regulatorofTcell activation (Crabtreeand Olson,2002;Hoganetal., 2003;Macian, 2005;Raoetal., 1997).”

“All NFAT proteins make very similar contacts with DNA but can have distinct expression patterns and functions as judged by the non-overlapping phenotypes ofmice deficient in individual NFAT family members (Crabtree and Olson, 2002; Hogan et al., 2003;Macian, 2005).”

“Because the RIT mutation does not interfere with NFAT:FOXP3 cooperation (data not shown), confirming predictions from the crystal structures that NFAT proteins make partially distinct contacts with Fos-Jun and FOXP proteins (Bandukwala et al., 2011; Chen et al., 1998; Wu et al., 2006), it might be possible to design r…”

“…its versatility, NFAT forms dimers on palindromic kB-like sequence elements and can bind DNA as a monomer (Chen et al., 1998; Giffin et al., 2003; Jin et al., 2010; Stroud et al., 2002); it also forms cooperative complexes with FOXP3, a transcription factor central to T cell regulatory function…”

“When differentiated into ‘‘memory’’ CTLs (Pipkin et al., 2010) and restimulated with PMA and ionomycin in vitro, NFAT1,2 double-deficient CD8+ T cells showed clearly decreased induction of the effector cytokines IL-2 and IFN-g compared toWT or singly NFAT1- or NFAT2-deficient CD8+ T cells (Figure 5A); they also showed…”

“Among these is the well-known composite NFAT:AP-1 element in the Il2 promoter (Figure 4B, left; Chen et al., 1998; Jain et al., 1992, 1993).”

“Moreover, antibodies to PD-1 and CTLA-4 have emerged as surprisingly effective agents for cancer immunotherapy (Schietinger and Greenberg, 2014), and individual or combined blockade of TIM3, PD-1, and LAG3 with antagonist antibodies has been shown to reverse exhaustion efficiently in CD8 T cells (Blackburn et al.”

“Fos and Jun are elevated only transiently after stimulation (Jain et al., 1992), whereas NFAT can remain in the nucleus for long times even in response to very low elevations of intracellular Ca2+ (Dolmetsch et al., 1997; Marangoni et al., 2013; Oh-Hora et al., 2008).”

“…in CD8+ and CD4+ T cells by CA-RIT-NFAT1 and those observed in exhausted CD8+ T cells and anergic CD4+ T cells in vivo (22/56 genes and 99/371 genes, respectively; p value < 10 10) (Figures 2E, 2F, and S3E; Tables 1, S1, and S2; Doering et al., 2012; Okamura et al., 2009; Wherry et al., 2007).”

“…can bind DNA as a monomer (Chen et al., 1998; Giffin et al., 2003; Jin et al., 2010; Stroud et al., 2002); it also forms cooperative complexes with FOXP3, a transcription factor central to T cell regulatory function (Bandukwala et al., 2011; Chen et al., 1998; Macián et al., 2000; Wuet al., 2006).”

“Thus, CA-RIT-NFAT1 expression affects two of the earliest steps of TCR signaling upstream of Ca entry; other steps in the signaling window between TCR stimulation and ER store depletion could potentially also be impaired (Heissmeyer et al., 2004).”

“We propose that this negative feedback program becomes dominant in CD4 T cells under conditions of ineffective costimulation (Mueller, 2010; Nurieva et al., 2011), and in CD8 T cells in the context of prolonged low-grade antigenic stimulation and/or inflammation, as encountered in chronic viral infections and cancer (…”

“P14+Tcra / mice were further crossed with NFAT1-deficient mice (Xanthoudakis et al., 1996) and with mice deficient in NFAT2 in the T cell lineage, obtained by breeding CD4Cre mice with mice in which exon 3 of the gene encoding NFAT2 was ‘‘floxed’’ (flanked with LoxP sites) (Figure S5A).”

“The residual expression was due to NFAT4, the third NFAT protein present in immune cells: NFAT1,2-deficient cells additionally transduced with shRNA against NFAT4 (Figure 5B) produced little or no IL-2 or interferon-g (IFN-g) upon restimulation (Figure 5A) and expressed little or no LAG3 and PD-1 either under resting…”

“…nuclear and therefore constitutively active (CA) (FigureS1A;Okamuraet al., 2000) andalsounable to interact with AP-1 (‘‘RIT’’ refers to three residues—R468, I469, and T535 inmouseNFAT1—that have beenmutated to interfere selectively with the NFAT:AP-1 interaction [Macián et al., 2000, 2002]).”

“In Vivo Tumor Model Single-cell suspensions from spleens and LNs of CL4 mice were pulsed with 10 mM HA515–523 peptide for 1 hr at 37 C, then cultured with 10 ng/ml murine rIL-12 for the first 2 days and 20 ng/ml murine rIL-2 for the following 5 days as previously described (Bauer et al., 2014).”

“, 2007]), or upregulated in anergic CD4CD25 CD45RBLAG3 T cells compared to CD4CD25 CD45RBLAG3 T cells (bottom) (Okamura et al., 2009).”

“…during chronic, high-dose LCMV infection in mice after 16 hr of stimulation (they did not examine other NFAT proteins), an alternative interpretation of their data is that the short inducible isoform of NFAT2 (Chuvpilo et al., 2002) is poorly induced after restimulation of exhausted CD8+ T cells.”

“mRNAs encoding E3 ligases (Fathman and Lineberry, 2007; Heissmeyer et al., 2004; Nurieva et al., 2011); phosphatases (Macián et al.”

“Fos and Jun are elevated only transiently after stimulation (Jain et al., 1992), whereas NFAT can remain in the nucleus for long times even in response to very low elevations of intracellular Ca2+ (Dolmetsch et al., 1997; Marangoni et al., 2013; Oh-Hora et al., 2008).”

“Fos and Jun are elevated only transiently after stimulation (Jain et al., 1992), whereas NFAT can remain in the nucleus for long times even in response to very low elevations of intracellular Ca2+ (Dolmetsch et al., 1997; Marangoni et al., 2013; Oh-Hora et al., 2008).”

“…not shown), confirming predictions from the crystal structures that NFAT proteins make partially distinct contacts with Fos-Jun and FOXP proteins (Bandukwala et al., 2011; Chen et al., 1998; Wu et al., 2006), it might be possible to design reagents that disrupt the NFAT:AP-1 inter- action but…”

“…with IRES-GFP retrovirus, empty (mock), or expressing either CA-RIT-NFAT1 or CA-RIT-NFAT1 with four point mutations in the DNA binding loop that abolish DNA binding (DBDmut-CA-RIT-NFAT1) (Jain et al., 1995); and expanded with interleukin-2 (IL-2) to generate effector CTLs (Pipkin et al., 2010).”

“Indeed, the higher basal Ca2+ concentrations observed by Agnellini et al. (2007) in exhausted CD8+ T cells and in CA-RITexpressing cells in this report would increase NFAT-dependent induction of NFAT2 under basal conditions, as also observed in ex vivo exhausted cells (Wherry et al., 2007).”

“Other pathways might also be involved: Notch (Mathieu et al., 2013) and type I IFN (Terawaki et al., 2011) signaling regulate Pdcd1 expression, and MEK kinase (Yoon et al., 2011) and T-bet (Anderson et al., 2010) regulate TIM3 expression.”

“Other pathways might also be involved: Notch (Mathieu et al., 2013) and type I IFN (Terawaki et al., 2011) signaling regulate Pdcd1 expression, and MEK kinase (Yoon et al., 2011) and T-bet (Anderson et al., 2010) regulate TIM3 expression.”

“Adding to its versatility, NFAT forms dimers on palindromic kB-like sequence elements and can bind DNA as a monomer (Chen et al., 1998; Giffin et al., 2003; Jin et al., 2010; Stroud et al., 2002); it also forms cooperative complexes with FOXP3, a transcription factor central to T cell regulatory function (Bandukwala e…”

“In contrast, signaling pathways regulating Ras, ERK, and AP-1 activation exhibit a shorter signal memory (Faroudi et al., 2003), suggesting that tolerance and/or exhaustion programs are evoked in tumor-infiltrating T cells as a result of an altered balance between NFAT NFAT1 in WT cells transduced…”

“Adding to its versatility, NFAT forms dimers on palindromic kB-like sequence elements and can bind DNA as a monomer (Chen et al., 1998; Giffin et al., 2003; Jin et al., 2010; Stroud et al., 2002); it also forms cooperative complexes with FOXP3, a transcription factor central to T cell regulatory…”

“, 2002), hyporesponsive states induced in T cells exposed to activating signals through the T cell receptor (TCR) in the absence of positive or presence of negative costimulatory signals (Nurieva et al., 2011).”

“…(Figure 4C) and no enrichment for kB-like sites such as the site in the Rnf128 promoter that binds NFAT1 homodimers (50- GTAACGTTTCC-30 or 50-GGATTCTTCC-30) (Soto-Nieves et al., 2009); Rnf128, which encodes the E3 ligase Grail, is upregulated in anergic CD4+ T cells (Fathman and Lineberry, 2007).”

“…E3 ligases (Fathman and Lineberry, 2007; Heissmeyer et al., 2004; Nurieva et al., 2011); phosphatases (Macián et al., 2002); and other signaling proteins including caspase 3, RGS proteins, and diacylglycerol kinases (Joshi and Koretzky, 2013; Macián et al., 2002) are upregulated in both cases.”

“We previously linked NFAT not only to T cell activation but also to T cell ‘‘tolerance’’ and ‘‘anergy’’ (Fehr et al., 2010; Heissmeyer et al., 2004; Macián et al., 2002), hyporesponsive states induced in T cells exposed to activating signals through the T cell receptor (TCR) in the absence of…”

“, 2007); NFAT2 was shown to be nuclear in tolerant CD8 T cells (Srinivasan and Frauwirth, 2007); and Pdcd1 expression was shown to be regulated by NFAT2 (Oestreich”

“Other pathways might also be involved: Notch (Mathieu et al., 2013) and type I IFN (Terawaki et al., 2011) signaling regulate Pdcd1 expression, and MEK kinase (Yoon et al., 2011) and T-bet (Anderson et al., 2010) regulate TIM3 expression.”

“Another variable gene of interest was the transcription factor NFATC1; previously implicated in T cell exhaustion (49).”

“NFAT is a family of transcription factors with well-established roles in T cell activation (261, 262), T cell anergy (263), and T cell exhaustion (7, 9, 122).”