Transient “rest” induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

“Gene set enrichment analysis was performed using the GSEA software (Broad Institute) as described (Mootha et al., 2003; Subramanian et al., 2005).”

“Gene set enrichment analysis was performed using the GSEA software (Broad Institute) as described (Mootha et al., 2003; Subramanian et al., 2005).”

“44) (Bray et al., 2016) index against hg38 reference genome.”

“PD-1 blockade can reinvigorate exhausted T cells (Barber et al., 2006) and therapeutic agents targeting the PD-1/PD-L1 axis have improved outcomes for many patients with cancer, but durable anti-tumor effects occur in only a fraction of those treated (Ribas and Wolchok, 2018).”

“ATAC-seq CAR-T cells were sorted on CD8 (see Cell Sorting) and processed for ATAC-seq, which was carried out as previously reported (Corces et al., 2017).”

“CAR-T cells mediate high response rates in relapsed/refractory high-grade B cell malignancies, but less than 50% of patients experience long-term disease control and CAR-T cells have not demonstrated reproducible efficacy against solid tumors (Davila et al., 2014; Lee et al., 2015; Majzner and Mackall, 2019; Maude et…”

“PD-1 blockade can reinvigorate exhausted T cells (Barber et al., 2006) and therapeutic agents targeting the PD-1/PD-L1 axis have improved outcomes for many patients with cancer, but durable anti-tumor effects occur in only a fraction of those treated (Ribas and Wolchok, 2018).”

“CAR-T cells mediate high response rates in relapsed/refractory high-grade B cell malignancies, but less than 50% of patients experience long-term disease control and CAR-T cells have not demonstrated reproducible efficacy against solid tumors (Davila et al., 2014; Lee et al., 2015; Majzner and Mackall, 2019; Maude et…”

“ATAC-seq CAR-T cells were sorted on CD8 (see Cell Sorting) and processed for ATAC-seq, which was carried out as previously reported (Corces et al., 2017).”

“CAR-T cells mediate high response rates in relapsed/refractory high-grade B cell malignancies, but less than 50% of patients experience long-term disease control and CAR-T cells have not demonstrated reproducible efficacy against solid tumors (Davila et al., 2014; Lee et al., 2015; Majzner and Mackall, 2019; Maude et…”

“Consistent with murine models of viral infection wherein antigen clearance induces T cell memory rather than exhaustion (Joshi et al., 2007; Rutishauser et al., 2009; Wherry et al., 2007; Wherry et al., 2004), early cessation of tonic CAR signaling (RestedD7-11) redirected T cell differentiation away from exhaustion a…”

“Several groups have sought to identify subsets of exhausted T cells that retain the capacity for reversal of the exhaustion program (Im et al., 2016; Miller et al., 2019; Utzschneider et al., 2016; Utzschneider et al., 2013).”

“Several groups have sought to identify subsets of exhausted T cells that retain the capacity for reversal of the exhaustion program (Im et al., 2016; Miller et al., 2019; Utzschneider et al., 2016; Utzschneider et al., 2013).”

“Chronic antigen stimulation induces T cell exhaustion, a differentiation state associated with a heritable epigenetic imprint that is distinct from that found in effector and memory T cells (Pauken et al., 2016; Philip et al., 2017; Schietinger et al., 2012; Schietinger et al., 2016; Sen et al., 2016; Wherry et al., 2…”

“RestedD7-11 cells also exhibited a sharp decline in T-bet and Blimp-1 expression levels and co-expression frequency (Figure S2F), consistent with transcriptional reprogramming of the population (Joshi et al., 2007; Rutishauser et al., 2009).”

“T cell exhaustion has been implicated as an important factor limiting the efficacy of CAR-T cells against cancer (Eyquem et al., 2017; Fraietta et al., 2018a; Fraietta et al., 2018b) and can be driven by excessive CAR signaling as a result of high antigen burden or tonic signaling induced by antigen-independent cluste…”

“We previously demonstrated that some CARs, like those incorporating a GD2-specific 14g2a scFv and CD28 co-stimulatory domain, undergo antigen-independent, tonic CAR signaling due to spontaneous receptor clustering, which promotes hallmark features of exhaustion in human T cells (Long et al., 2015; Lynn et al., 2019).”

“T cell exhaustion has been implicated as an important factor limiting the efficacy of CAR-T cells against cancer (Eyquem et al., 2017; Fraietta et al., 2018a; Fraietta et al., 2018b) and can be driven by excessive CAR signaling as a result of high antigen burden or tonic signaling induced by…”

“ChromVAR was used to compute the relative differential accessibility of known transcription factor motifs across different samples as previously described (Rubin et al., 2019; Schep et al., 2017).”

“Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of human T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation (Alfei et al., 2019; Khan et al., 2019; Scott et al., 2019; Seo et al., 2019; Wang et al., 2019; Yao et al., 2019).”

“…exhaustion, a differentiation state associated with a heritable epigenetic imprint that is distinct from that found in effector and memory T cells (Pauken et al., 2016; Philip et al., 2017; Schietinger et al., 2012; Schietinger et al., 2016; Sen et al., 2016; Wherry et al., 2007; Zajac et al.,…”

“A merged peak list was created as previously described(Corces et al., 2018).”

“Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of human T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation (Alfei et al., 2019; Khan et al., 2019; Scott et al., 2019; Seo et al., 2019; Wang et al., 2019; Yao et al., 2019).”

“Chimeric antigen receptors (CARs) combine a tumor antigen-recognition domain with intracellular signaling domains, thereby enabling recognition and killing of tumor cells in a major histocompatibility complex (MHC)-independent manner (Lim and June, 2017; Mackall et al., 2014).”

“Several groups have sought to identify subsets of exhausted T cells that retain the capacity for reversal of the exhaustion program (Im et al., 2016; Miller et al., 2019; Utzschneider et al., 2016; Utzschneider et al., 2013).”

“Chronic antigen stimulation induces T cell exhaustion, a differentiation state associated with a heritable epigenetic imprint that is distinct from that found in effector and memory T cells (Pauken et al., 2016; Philip et al., 2017; Schietinger et al., 2012; Schietinger et al., 2016; Sen et al., 2016; Wherry et al., 2…”

“Similarly, murine tumor models have revealed that exhaustion reprogrammability is associated with increased accessibility at the Tcf7 locus (Philip et al., 2017).”

“Using a drug-regulatable platform (Banaszynski et al., 2006; Banaszynski et al., 2008) wherein a tonically signaling CAR was modified with a C-terminal destabilizing domain (DD) to enable drug-dependent control of CAR protein levels, we observed that transient inhibition of CAR surface expression,…”

“Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of human T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation (Alfei et al., 2019; Khan et al., 2019; Scott et al., 2019; Seo et al., 2019; Wang et al., 2019; Yao et al., 2019).”

“…modified a validated in vitro model of CAR-associated T cell exhaustion, wherein tonic CAR signaling induces hallmark phenotypic, functional, transcriptomic and epigenetic features of exhaustion within 11 days (HA.28ζ) (Lynn et al., 2019), to enable precise, drug-dependent control of CAR signaling.”

“…which is associated with a progenitor exhausted cell population that retains anti-tumor functionality and is responsive to checkpoint blockade (Chen et al., 2019c; Im et al., 2016; Jadhav et al., 2019; Utzschneider et al., 2016), was similar in both Always ON and RestedD7-11 conditions and…”

“T cell exhaustion has been implicated as an important factor limiting the efficacy of CAR-T cells against cancer (Eyquem et al., 2017; Fraietta et al., 2018a; Fraietta et al., 2018b) and can be driven by excessive CAR signaling as a result of high antigen burden or tonic signaling induced by…”

“12 AP-1 family TF motifs (BATF, BATF3, JUNB, NFATC2), which are known to promote T cell exhaustion (Kurachi et al., 2014; Lynn et al., 2019; Man et al., 2017; Martinez et al., 2015; Scott-Browne et al., 2016), were highly enriched in accessible regions of the Always ON epigenome, but rendered less accessible following…”

“Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of human T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation (Alfei et al., 2019; Khan et al., 2019; Scott et al., 2019; Seo et al., 2019; Wang et al., 2019; Yao et al., 2019).”

“QUANTIFICATION AND STATISTICAL ANALYSES Mass cytometry data processing and analysis Acquired data was normalized and debarcoded using MATLAB-based software (Finck et al., 2013; Zunder et al., 2015).”

“Chronic antigen stimulation induces T cell exhaustion, a differentiation state associated with a heritable epigenetic imprint that is distinct from that found in effector and memory T cells (Pauken et al., 2016; Philip et al., 2017; Schietinger et al., 2012; Schietinger et al., 2016; Sen et al., 2016; Wherry et al., 2…”

“…which is associated with a progenitor exhausted cell population that retains anti-tumor functionality and is responsive to checkpoint blockade (Chen et al., 2019c; Im et al., 2016; Jadhav et al., 2019; Utzschneider et al., 2016), was similar in both Always ON and RestedD7-11 conditions and…”

“Advances in synthetic biology have enabled the development of several tunable CAR platforms which have been optimized to mitigate CAR-mediated toxicity (Bejestani et al., 2017; Cho et al., 2018; Leung et al., 2019; Rodgers et al., 2016; Wu et al., 2015).”

“Advances in synthetic biology have enabled the development of several tunable CAR platforms which have been optimized to mitigate CAR-mediated toxicity (Bejestani et al., 2017; Cho et al., 2018; Leung et al., 2019; Rodgers et al., 2016; Wu et al., 2015).”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“Since PD-1 and PD-L1 are both expressed on activated T cells (Latchman et al., 2004) (Figure S2E), in some experiments we included a condition wherein Always ON T cells were cultured with aPD-1-blocking antibody from D7-15 to compare the degree of reprogramming induced by checkpoint blockade with that of transient res…”

“RestedD7-11 cells also exhibited a sharp decline in T-bet and Blimp-1 expression levels and co-expression frequency (Figure S2F), consistent with transcriptional reprogramming of the population (Joshi et al., 2007; Rutishauser et al., 2009).”

“QUANTIFICATION AND STATISTICAL ANALYSES Mass cytometry data processing and analysis Acquired data was normalized and debarcoded using MATLAB-based software (Finck et al., 2013; Zunder et al., 2015).”

“Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of human T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation (Alfei et al., 2019; Khan et al., 2019; Scott et al., 2019; Seo et al., 2019; Wang et al., 2019; Yao et al., 2019).”

“Reinvigoration of exhausted CAR-T cells using the Src kinase inhibitor dasatinib We and others recently demonstrated that dasatinib, an FDA-approved tyrosine kinase inhibitor, suppresses CAR-T cell activation via rapid and reversible antagonism of proximal T cell receptor (TCR) signaling kinases (Mestermann et al., 20…”

“Consistent with murine models of viral infection wherein antigen clearance induces T cell memory rather than exhaustion (Joshi et al., 2007; Rutishauser et al., 2009; Wherry et al., 2007; Wherry et al., 2004), early cessation of tonic CAR signaling (RestedD7-11) redirected T cell differentiation away from exhaustion a…”

“AP-1 family TF motifs (BATF, BATF3, JUNB, NFATC2), which are known to promote T cell exhaustion (Kurachi et al., 2014; Lynn et al., 2019; Man et al., 2017; Martinez et al., 2015; Scott-Browne et al., 2016), were highly enriched in accessible regions of the Always ON epigenome, but rendered less…”

“Advances in synthetic biology have enabled the development of several tunable CAR platforms which have been optimized to mitigate CAR-mediated toxicity (Bejestani et al., 2017; Cho et al., 2018; Leung et al., 2019; Rodgers et al., 2016; Wu et al., 2015).”

“To interrogate DD-CAR functionality in vivo, we substituted the FKBP DD for an E. coli-derived dihydrofolate reductase DD (ecDHFR) (Iwamoto et al., 2010).”

“AP-1 family TF motifs (BATF, BATF3, JUNB, NFATC2), which are known to promote T cell exhaustion (Kurachi et al., 2014; Lynn et al., 2019; Man et al., 2017; Martinez et al., 2015; Scott-Browne et al., 2016), were highly enriched in accessible regions of the Always ON epigenome, but rendered less…”

“Recent studies suggest that the epigenetic modifier, enhancer of zeste homolog 2 (EZH2), which catalyzes trimethylation on histone 3 at lysine 27 (H3K27me3) as a subunit of the polycomb repressive complex 2 (PRC2), prevents hematopoietic stem cell exhaustion and is critical for T cell differentiation, maintenance of T…”

“…JUNB, NFATC2), which are known to promote T cell exhaustion (Kurachi et al., 2014; Lynn et al., 2019; Man et al., 2017; Martinez et al., 2015; Scott-Browne et al., 2016), were highly enriched in accessible regions of the Always ON epigenome, but rendered less accessible following rest…”

“We observed similar phenotypic and functional reinvigoration following transient exposure to dasatinib, a Src kinase inhibitor that potently and reversibly inhibits TCR and CAR signaling (Lee et al., 2010; Mestermann et al., 2019; Schade et al., 2007; Weber et al., 2019).”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of human T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation (Alfei et al., 2019; Khan et al., 2019; Scott et al., 2019; Seo et al., 2019; Wang et al., 2019; Yao et al., 2019).”

“Several groups have sought to identify subsets of exhausted T cells that retain the capacity for reversal of the exhaustion program (Im et al., 2016; Miller et al., 2019; Utzschneider et al., 2016; Utzschneider et al., 2013).”

“Reinvigoration of exhausted CAR-T cells using the Src kinase inhibitor dasatinib We and others recently demonstrated that dasatinib, an FDA-approved tyrosine kinase inhibitor, suppresses CAR-T cell activation via rapid and reversible antagonism of proximal T cell receptor (TCR) signaling kinases (Mestermann et al., 20…”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“Clonotypes were further assessed using tcR, which enabled computation of clontype abundance and diversity estimations based on the CDR3 amino acid length and V gene usage (Nazarov et al., 2015).”

“Indeed, improved antigen-specific T cell function has been associated with antigen clearance in humans with hepatitis C infection treated with direct acting anti-viral therapies (Burchill et al., 2015; Kohli et al., 2015; Martin et al., 2014; Shrivastava et al., 2017; Wieland et al., 2017).”

“Indeed, improved antigen-specific T cell function has been associated with antigen clearance in humans with hepatitis C infection treated with direct acting anti-viral therapies (Burchill et al., 2015; Kohli et al., 2015; Martin et al., 2014; Shrivastava et al., 2017; Wieland et al., 2017).”

“…exhausted cell population that retains anti-tumor functionality and is responsive to checkpoint blockade (Chen et al., 2019c; Im et al., 2016; Jadhav et al., 2019; Utzschneider et al., 2016), was similar in both Always ON and RestedD7-11 conditions and represented approximately 10% of total…”

“…a differentiation state associated with a heritable epigenetic imprint that is distinct from that found in effector and memory T cells (Pauken et al., 2016; Philip et al., 2017; Schietinger et al., 2012; Schietinger et al., 2016; Sen et al., 2016; Wherry et al., 2007; Zajac et al., 1998).”

“Indeed, improved antigen-specific T cell function has been associated with antigen clearance in humans with hepatitis C infection treated with direct acting anti-viral therapies (Burchill et al., 2015; Kohli et al., 2015; Martin et al., 2014; Shrivastava et al., 2017; Wieland et al., 2017).”

“Using a drug-regulatable platform (Banaszynski et al., 2006; Banaszynski et al., 2008) wherein a tonically signaling CAR was modified with a C-terminal destabilizing domain (DD) to enable drug-dependent control of CAR protein levels, we observed that transient inhibition of CAR surface expression,…”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“TF binding motifs for genes that have been implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family) (Hu and Chen, 2013; Jadhav et al., 2019; Kratchmarov et al., 2018; Michelini et al., 2013; Milner et al., 2017; Rao et al., 2012; Sullivan et al., 2012;…”

“We observed similar phenotypic and functional reinvigoration following transient exposure to dasatinib, a Src kinase inhibitor that potently and reversibly inhibits TCR and CAR signaling (Lee et al., 2010; Mestermann et al., 2019; Schade et al., 2007; Weber et al., 2019).”

“Chimeric antigen receptors (CARs) combine a tumor antigen-recognition domain with intracellular signaling domains, thereby enabling recognition and killing of tumor cells in a major histocompatibility complex (MHC)-independent manner (Lim and June, 2017; Mackall et al., 2014).”

“Advances in synthetic biology have enabled the development of several tunable CAR platforms which have been optimized to mitigate CAR-mediated toxicity (Bejestani et al., 2017; Cho et al., 2018; Leung et al., 2019; Rodgers et al., 2016; Wu et al., 2015).”

“**, p<0.01; ****, p<0.0001; ns, p>0.05 Unbiased multidimensional analyses using force-directed layouts (FDLs) (Good et al., 2019; Samusik et al., 2016), which map phenotypically similar cells closely together and dissimilar cells farther apart, illustrated phenotypic trajectories towards exhaustion…”

“…which is associated with a progenitor exhausted cell population that retains anti-tumor functionality and is responsive to checkpoint blockade (Chen et al., 2019c; Im et al., 2016; Jadhav et al., 2019; Utzschneider et al., 2016), was similar in both Always ON and RestedD7-11 conditions and…”

“Similarly, an immunomodulatory effect of dasatinib on T cells has been associated with improved anti-tumor immunity (Hekim et al., 2017).”

“We previously demonstrated that some CARs, like those incorporating a GD2-specific 14g2a scFv and CD28 co-stimulatory domain, undergo antigen-independent, tonic CAR signaling due to spontaneous receptor clustering, which promotes hallmark features of exhaustion in human T cells (Long et al., 2015; Lynn et al., 2019).”

“…which is associated with a progenitor exhausted cell population that retains anti-tumor functionality and is responsive to checkpoint blockade (Chen et al., 2019c; Im et al., 2016; Jadhav et al., 2019; Utzschneider et al., 2016), was similar in both Always ON and RestedD7-11 conditions and…”

“Finally, transient disruption of tonic CAR signaling via regulation of CAR protein can epigenetically repro- 54 Cell 181, April 2, 2020 gram exhausted CAR T cells and augment efficacy in pre-clinical models (Weber et al., 2020).”

“Using dasatinib or a destabilization domain, transient rest from CAR signaling protects against T cell exhaustion ( 50 ); future work may establish whether lenalidomide ON-or OFF-switch control enhances the phenotype and function of exhaustion-prone CARs.”

“These results provide clear evidence that SNIP CARs are biologically distinct and more potent than constitutive CARs, which we posit results from variability in GPV levels associated with normal PK that oscillate CAR signaling, thereby preventing exhaustion and improving CAR-T cell function (Weber et al., 2020).”

“…reported that human HA-28z CAR T cells, targeting the GD2 surface marker, exhibit tonic signaling and lead to an exhausted T cell phenotype following 10 days of transduction, in contrast to the non-exhausted phenotype of human CD19-28z CAR T cells, targeting the CD19 surface marker 7,16 .”

“As strategies emerge to control the expression of CARs (29, 34) as well as modulate TCR/CAR signaling (28, 60, 61), our findings reinforce the potential for strategies to mitigate T cell exhaustion in the TME by regulating TCR/CAR expression and/or signaling, thereby augmenting the therapeutic efficacy of adoptive T c…”

“This state of functional exhaustion can be manipulated by limiting activity of signaling pathways, and altering the epigenetic program and/or metabolic pathways (Chen et al., 2019; Fraietta et al., 2018; Lynn et al., 2019; Weber et al., 2020b).”

“One reason of failure is T cell dysfunction, due to T cell exhaustion [2, 7, 8].”