Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling

1Chimeric antigen receptors (CARs) combine a tumor antigen-recognition domain with intracellular signaling domains, enabling recognition and killing of tumor cells in a major histocompatibility complex (MHC)-independent manner. 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. T cell exhaustion has been implicated as an important factor limiting the efficacy of CAR-T cells against cancer and can be driven by excessive CAR signaling as a result of high antigen burden or tonic signaling induced by antigen-independent clustering of the CAR receptor. We hypothesized that transient cessation of CAR signaling would enable exhausted CAR-T cells to regain functionality and form a memory pool, similar to the effects observed following antigen clearance of acute infections.

2Using a drug-regulatable platform 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 (“rest”), and thereby tonic CAR signaling, prevented cells from developing phenotypic hallmarks of exhaustion, instead redirecting them to a memory-like fate. Furthermore, transient rest, but not PD-1 blockade, restored anti-tumor functionality in a cell population that had already acquired functional, transcriptional and epigenetic features of exhaustion, and was associated with global transcriptional and epigenetic reprogramming. We observed similar phenotypic and functional reinvigoration following transient exposure to dasatinib, a tyrosine kinase inhibitor that reversibly inhibits TCR and CAR signaling. Collectively, these results challenge the concept that exhaustion is an epigenetically fixed state and reveal that transient cessation of CAR signaling may provide a strategy for augmenting function of exhausted human CAR-T cell populations.

3We previously demonstrated that some CARs undergo antigen-independent, tonic CAR signaling due to spontaneous receptor clustering, which promotes hallmark features of exhaustion in human T cells. To test whether prevention of tonic signaling preserves CAR-T cell functionality, we incorporated an FK506 binding protein 12 (FKBP) destabilizing domain (DD) into a tonically signaling GD2-targeting CAR (GD2.28ζ.FKBP), similar to a recently published study. The DD induced rapid degradation of the CAR at baseline (Fig. 1, A and B), whereas shield-1, which stabilizes the FKBP DD, prevented CAR degradation thereby inducing GD2.28ζ.FKBP CAR expression on the cell surface in a dose- and time-dependent manner (Fig. 1, B and C). Removal of shield-1 led to a rapid decrease in CAR surface protein, with a degradation half-life of approximately 1 hour (Fig. 1C). To determine whether the dynamic range of CAR expression induced by the DD modulated biologic reactivity and to assess the relationship between CAR expression level and function, we co-cultured GD2.28ζ.FKBP CAR-T cells with Nalm6 leukemia engineered to express GD2, GFP, and luciferase (Nalm6-GD2) with increasing concentrations of shield-1. We observed drug-dependent, tunable control of tumor-induced cytokine secretion (Fig. 1D) and cytotoxicity (Fig. 1E), demonstrating that modulation of CAR expression levels tuned CAR-T cell function.

4To interrogate DD-CAR functionality in vivo, we engineered a GD2.28ζ CAR incorporating an E. coli-derived dihydrofolate reductase DD (GD2.28ζ.ecDHFR), which is regulated by the FDA-approved antibiotic, trimethoprim (TMP), and demonstrated a dynamic range comparable to that of the GD2.28ζ.FKBP CAR (fig. S1A). In NOD/SCID/IL2Rγ−/− (NSG) mice engrafted with Nalm6-GD2 leukemia, intraperitoneal TMP administration upregulated surface CAR and CD69 in CAR-T cells isolated from blood and spleen compared to controls (fig. S1B). Together, these data demonstrate drug-dependent control of DD-CAR expression and activity in vitro and in vivo.

5We next sought to determine whether control of DD-CAR expression prevented exhaustion induced by tonic CAR signaling. GD2.28ζ.FKBP CAR-T cells cultured with shield-1 (ON) exhibited antigen-independent phosphorylation of CAR CD3ζ (CD3ζ) (fig. S1C) and elevated expression of inhibitory receptors PD-1, TIM-3, and LAG-3 (fig. S1D), compared to those cultured without shield-1 (OFF). GD2.28ζ.FKBP or GD2.28ζ.ecDHFR T cells expanded in the OFF state, but provided drug just prior to antigen challenge (OFF/ON) exhibited superior cytotoxicity (fig. S1E), tumor-induced cytokine secretion (fig. S1F), and enhanced efficacy and cell expansion in vivo (Fig. 1, F and G). Collectively, these observations confirm that tonic CAR signaling induces T cell dysfunction and demonstrate that prevention of tonic signaling ex vivo augments CAR-T cell efficacy.

6We next regulated CAR expression in HA.28ζ-CAR-T cells (fig. S2A), which manifest extremely robust tonic signaling and acquire functional, transcriptomic and epigenetic hallmarks of exhaustion by D11 (8). When compared to HA.28ζ.FKBP CAR-T cells continuously cultured with shield-1 (Always ON), cells from which shield-1 was removed on D7 (RestedD7-11) demonstrated decreased inhibitory receptor expression and superior functionality upon tumor challenge on D11 (Fig. 2, A through C). Mass cytometric analysis of 27 proteins associated with T cell exhaustion, activation, or memory (fig. S2B, Table S1) demonstrated that CD8+ Always ON cells manifested time-dependent increases in exhaustion scores (normalized mean expression of PD-1, TIM-3, LAG-3, CTLA-4, BTLA, 2B4, and CD39), whereas RestedD7-11 cells demonstrated time-dependent increases in memory scores (normalized mean expression of CD45RA, IL-7R, CD27, CD197) (Fig. 2D).

7Force-directed layouts (FDLs), which map phenotypically similar cells closely together and dissimilar cells farther apart, illustrated trajectories towards exhaustion or memory-like cell fates. CD8+ Always ON and RestedD7-11 CAR-T cells displayed substantial evolution during D7-11 and disparate phenotypes by D11 were spatially distributed between 4 distinct regions on the FDL (Fig. 2E and fig. S2C). The majority of D11 Always ON cells resided in Regions 1 or 2, while the majority of RestedD7-11 cells resided in Regions 3 or 4 (Fig. 2F). FDLs also revealed heterogeneity within the exhausted and memory subsets, with Region 1 demonstrating a higher exhaustion score and increased CD39 compared to Region 2 (fig. S2D), and Region 4 demonstrating a higher memory score and increased expression of IL7RA and CD45RA compared to Region 3 (Fig. 2, G and H, and fig. S2E). RestedD7-11 cells exhibited reduced T-bet and Blimp-1 expression levels and co-expression frequency (fig. S2F), consistent with transcriptional reprogramming.

8Redirection of cell fate induced by rest from D7-11 was most likely due to population-wide changes rather than outgrowth of a rare subpopulation since we detected only modest differences in fold change in cell expansion, Ki-67 expression, and cleaved PARP (cPARP) between Always ON and RestedD7-11 cells (fig. S2, G and H). Moreover, TCF1 expression, which is associated with a progenitor exhausted cell population that retains anti-tumor functionality and is responsive to checkpoint blockade, was similar in Always ON and RestedD7-11 conditions, representing approximately 10% of total CD8+ CAR-T cells on D11 (fig. S2I). RNA-sequencing at D11 demonstrated that both Always ON and RestedD7-11 cells contained ~1,000 unique TCR clonotypes with similar clonotypic diversity (fig. S2, J and K). Similar phenotypic changes were observed in RestedD7-11 CD4+ CAR-T cells (fig. S3). Collectively, these observations indicate that transient cessation of CAR signaling prior to exhaustion onset alters the differentiation trajectory of a large fraction of CAR-T cell populations rather than inducing outgrowth of a minor subset of highly proliferative, apoptosis-resistant, or TCF1+ progenitor exhausted RestedD7-11 cells.

9Since cessation of tonic signaling altered CAR-T cell fate during the transition to exhaustion, we hypothesized that rest could also reprogram T cell populations on which exhaustion is already imprinted. We compared D15 Always ON HA.28ζ.FKBP CAR-T cells to cells rested from D11-15 (RestedD11-15, Fig. 3A). PD-1 and PD-L1 are both expressed on activated T cells (fig. S2E), and in some experiments we cultured Always ON T cells with αPD-1 from D7-15 to compare the effects of checkpoint blockade with that of transient rest. As expected, D15 Always ON cells exhibited robust tonic CAR signaling, poor expansion, elevated immune checkpoint receptors (PD-1, TIM-3 and LAG-3), and an effector-like phenotype (Fig. 3, B through E, and fig. S4, A through F) compared to Always OFF cells. In contrast, CD8+ RestedD11-15 cells displayed diminished tonic signaling, reduction in inhibitory receptor expression, increased stem cell memory-like cells (CD45RO−, CCR7+), and enhanced proliferation compared to Always ON and Always ON + αPD-1 T cells. Sequential RNA sequencing demonstrated that transcripts associated with exhaustion underwent rapid and complete reversal to baseline Always OFF levels in rested cells by D11 or D15, whereas αPD-1 in Always ON T cell cultures induced only a small but detectable effect on gene expression (Fig. 3F and fig. S4G).

10Unbiased principal component analysis (PCA) at each timepoint displayed overlap between Always OFF and rested conditions, which separated from Always ON and Always ON + αPD-1 cells along PC1 (Fig. 3G and fig. S4H), illustrating the high degree of transcriptomic reprogramming induced by rest. Hierarchical clustering of the top 500 genes driving PC1 variance identified exhaustion- (PDCD1, ENTPD1, BATF, NR4A1) and memory-associated genes (IL7R, LEF1, KLF2, BACH2)(Fig. 3H). D15 analyses confirmed transcriptomic reversal, since RestedD11-15 T cells significantly upregulated memory/quiescence-associated genes (SELL, LEF1, FOXO3) and downregulated canonical exhaustion-associated genes (CTLA-4, IRF4, TOX2, NR4A3) compared to Always ON T cells (p<0.05, fig. S4, I and J). Importantly, both exhausted and rested populations on D15 contained ~1,000-3,000 unique TCR clonotypes and exhibited similar clonotypic diversity and rates of apoptosis (fig. S4, K through M). These data are consistent with rest-induced changes occurring broadly within the population under study, rather than preferential expansion of a small subset of cells, which would result in reduced clonotypic diversity.

11We next tested whether the phenotypic and transcriptomic reprogramming of exhausted CAR-T cells induced by rest would confer enhanced anti-tumor functionality. Prior to antigen challenge, we treated CAR-T cells with shield-1 for 16 hours to normalize CAR surface expression (Fig. 4A). On D15, Rested7-15 and Rested11-15 T cells demonstrated marked enhancement in cytotoxicity and cytokine secretion compared to exhausted Always ON cells (Fig. 4, B and C, and fig. S5A). Single cell analyses revealed that approximately 60% of Always OFF and rested CAR-T cells were polyfunctional and capable of secreting at least two cytokines, whereas less than 20% of Always ON T cells exhibited these features (Fig. 4, D and E, and fig. S5, B and C), demonstrating functional reinvigoration in a high fraction of rested T cells. In contrast to rest, PD-1 blockade enhanced cytotoxicity but did not significantly augment IL-2 or IFNγ secretion, the frequency of cytokine-secreting cells, or sensitivity to low antigen (Fig. 4, B through G, and fig. S5, B and C), indicating that rest-associated functional reinvigoration is mechanistically distinct from that of checkpoint blockade. In vivo studies corroborated these findings (Fig. 4H and fig. S5, D and E), since exhausted Always ON T cells failed to control tumor growth, whereas RestedD7-15, and RestedD11-15 T cells cured or maintained the tumor at levels comparable to Always OFF (Fig. 4I, and fig. S5F). These data demonstrate that transient cessation of CAR signaling reverses the exhaustion phenotype and rescues CAR-T cell functionality.

12To determine the impact of transient rest on the epigenome, we used ATAC-seq to analyze differences in chromatin accessibility between Always ON, Always OFF, Rested7-15 and Rested11-15 HA.28ζ.FKBP CD8+ CAR-T cells (Fig. 5A and fig. S6A). Temporal analyses revealed that T cells experiencing continuous tonic CAR signaling (Always ON) were epigenetically distinct from activated CD19.28ζ CAR-T cells (fig. S6, B and C) and displayed dramatic alterations in chromatin accessibility within the first 7 days (~48,000 peak changes), with fewer changes occurring between D7-11 (~2,300 peak changes) and D11-15 (~2,000 peak changes) (Fig. 5B and fig. S6D). Notably, a 4-day rest period between D7-11 (RestedD7-11) or D11-15 (RestedD11-15) was associated with ~19,000 and ~15,500 peak changes, respectively (Fig. 5B), many of which were differentially accessible compared to Always ON cells (fig. S6E). These included exhaustion-associated genes ENTP1 and BATF, and the stemness-associated gene TCF7 (Fig. 5C). These results align with our observations of rapid phenotypic, functional and transcriptional reprogramming following 4 days of rest.

13Unbiased Pearson correlation and PCA analyses indicated that rest resulted in global, wholescale remodeling of the epigenome. Always ON cells treated with or without αPD-1 cluster together along PC1 on D11 (66.51% variance) and D15 (58% variance) and exhibit highly correlated genomic accessibility profiles (Fig. 5D and fig. S6, F and G). In contrast, Rested CAR-T cells exhibit clear separation from Always ON and cluster together with Always OFF cells along PC1 (Fig. 5D and fig. S6G). Binding motifs for AP-1 family TFs (BATF, JUNB, NFATC2), which promote T cell exhaustion, were enriched in accessible regions of the Always ON epigenome, but less accessible following rest (Fig. 5E and fig. S6H). TF binding motifs for many genes implicated in T cell memory were exclusively accessible in rested conditions (TCF7/TCF7L2, LEF1, RUNX family, FOXO family), whereas genes associated with T cell exhaustion (EOMES, TBX21) showed exaggerated inaccessibility compared to Always OFF cells (Fig. 5E), raising the prospect that T cell rest could induce a distinct program to drive the development of memory-like T cells. Biological processes inferred from D15 differentially accessible peaks revealed that rest is associated with telomere packaging and G1 phase, diminished Akt signaling, apoptosis, and negative regulation of Wnt signaling (fig. S6H), consistent with induction of a quiescent/memory T cell phenotype.

14The epigenetic modifier, enhancer of zeste homolog 2 (EZH2), which catalyzes trimethylation on histone 3 at lysine 27 (H3K27me3) as part of the polycomb repressive complex 2 (PRC2), prevents hematopoietic stem cell exhaustion and is critical for T cell differentiation, maintenance of T cell memory, and antitumor immunity. Unbiased PCA of H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) separated Always ON and Always OFF or RestedD11-15 CAR-T cells along PC2 (Fig. 5F), similar to RNA- and ATAC-seq data (Figs. 3G and 5D), indicating altered H3K27me3 in exhaustion. To test whether EZH2 contributes to reversal of exhaustion in this model, we rested Always ON exhausted CAR-T cells in the presence of the selective EZH2 inhibitor tazemetostat (EZH2i) from D11-15. As expected, EZH2i treatment reduced H3K27me3 levels in all conditions, but preferentially altered the H3K27me3 landscape in RestedD11-15 cells (Fig. 5, F and G, fig. S7A). Genomic regions containing decreased H3K27me3 levels in EZH2i-treated RestedD11-15 cells occurred near or within exhaustion-associated genes that undergo changes in accessibility during rest, including TBX21, NFATC1, AP-1 family TFs (FOS, FOSB, JUNB), as well as other known regulators of the epigenetic state of exhausted T cells (DMNT3A and NR4A family TFs) (Fig. 5H and fig. S7B).

15In contrast to inhibitors of DNA methyltransferases and histone acetyltransferases, which strongly attenuated anti-tumor function in all conditions, EZH2i induced only small but detectable effects on Always OFF T cell functionality (fig. S7C) while strongly and dose-dependently attenuating rescue of IL-2 secretion and tumor killing in RestedD11-15 CAR-T cells compared to vehicle-treated control. These results demonstrate a requirement for active chromatin remodeling during rest-associated reinvigoration of exhausted CAR-T cells (Fig. 5, I through K). Notably, EZH2i did not affect CAR-T cell viability, proliferation, or other functions (fig. S7, D through H), nor did it alter rest-associated reversal of the cell surface phenotype (fig. S7I), consistent with the observation that the epigenetic mechanisms governing exhausted T cell phenotype and dysfunction are distinct. Collectively, these data suggest that CAR-T cell rest remodels the exhaustion-associated epigenome through EZH2, thereby promoting functional reinvigoration.

16We and others recently demonstrated that dasatinib (Das), an FDA-approved tyrosine kinase inhibitor, suppresses CAR-T cell activation via rapid and reversible antagonism of proximal T cell receptor (TCR) signaling kinases. Consistent with this, dasatinib-treated HA.28ζ CAR-T cells exhibited undetectable phosphorylation of CAR CD3ζ and ERK1/2 compared to those treated with vehicle (Fig. S8A). We hypothesized that dasatinib-mediated inhibition of tonic CAR signaling could induce rest and reverse exhaustion. Indeed, HA.28ζ CAR-T cells treated with dasatinib exhibited improved expansion, diminished inhibitory receptor expression, functional reinvigoration (fig. S8, B through D), and improved tumor control following adoptive transfer (Fig. 6A). Ex vivo dasatinib treatment of CAR-T cells expressing GD2.BBζ, which exhibits a lesser degree of tonic signaling, also promoted a more memory-like phenotype and enhanced in vivo functionality (fig. S8, E and F), indicating that dasatinib provides an approach to mitigate deleterious CAR signaling in pre-clinical or clinical CAR-T cell manufacturing settings.

17We observed in these experiments that DasD11-15 T cells did not demonstrate equivalent anti-tumor functionality to DasD4-15 and DasD7-15 cells. To determine whether DasD11-15 cells encountered a point of irreversibility in this model system, or whether this reflected an inadequate period of rest to restore functionality, we interrogated exhaustion reversibility using a more protracted in vitro time course, resting in increments of 3-4 days from D4 until D25 (Fig. 6B). Since dasatinib completely suppresses CAR-T cell signaling and function (fig. S8A), whereas DD-CARs in the OFF state exhibited some leakiness in expression and function (Fig. 1 and fig. S1B), we opted to utilize dasatinib to induce rest in the protracted model system. All groups of D25 dasatinib-treated HA.28ζ CAR-T cells demonstrated diminished exhaustion marker expression and increased stem cell memory-associated CD62L and CD45RA expression (Fig. 6C and fig. S8G), diminished expression of exhaustion associated TFs T-bet and TOX, and increased expression of stemness-associated TFs LEF1 and TCF1 (Fig. 6D and fig. S8H), corroborating D15 epigenetic changes (Fig. 5, C and E). Dasatinib also rescued CAR-T cell cytotoxicity independent of treatment duration (Fig. 5E); however, complete rescue of antigen-induced cytokine secretion only occurred in groups with more prolonged dasatinib exposure (Figure 6F), consistent with a model wherein the incomplete reinvigoration observed on D15 (Fig. 6A and fig. S8D) reflected an insufficient period of rest rather than irreversibility of the exhaustion program (Fig. 6, F through G). This model is further corroborated by the correlation observed between the degree of functional reinvigoration and duration of rest, which was independent of the time at which rest was initiated (Fig. 6G and fig. S8, I through K). Initiation of rest at D29 or as late as D46 induced partial reversal of phenotypic and functional exhaustion hallmarks on D53, and was associated with decreased T-bet and TOX expression, indicating marked plasticity in the exhaustion program even at very late time points (Fig. 6, H and I, and fig. S9).

18Adoptive transfer of rested CAR-T cells induced more durable anti-tumor responses than exhausted CAR-T cells (Figs. 4I and 6A), suggesting that rested cells might resist reacquisition of the exhaustion program upon reactivation. To test this hypothesis, HA.28ζ exhausted CAR-T cells were “pulsed” with dasatinib for 4 days, then dasatinib was removed for 3 days prior to analysis (fig. S10A). CAR-T cells pulsed with dasatinib exhibited improved CD8+ expansion, diminished exhaustion marker expression, and enhanced functionality compared to HA.28ζ CAR-T cells treated with vehicle (fig. S10, B through E). Similar results were observed by toggling HA.28ζ.FKBP CAR surface expression (fig. S10, F through J), suggesting that repeated rest periods may prolong responses in vivo.

19To test whether intermittent rest results in more durable anti-tumor responses, mice engrafted with dasatinib-insensitive liquid (Nalm6 leukemia) or solid (143B osteosarcoma) tumors were infused with CAR-T cells and repeatedly pulsed with dasatinib or vehicle. Nalm6-bearing mice infused with a limiting dose of CD19.BBζ CAR-T cells and pulsed with dasatinib using a 3-day dasatinib/4-day off schedule exhibited more durable anti-tumor responses and improved survival compared to mice treated with vehicle or dasatinib every other day (Fig. 7, A and B, and fig. S11, A and B), further corroborating in vitro data showing that the duration of rest correlates with the degree of exhaustion reversal (Fig. 4C and Fig. 6, A through G). Similar results were achieved in 143B-bearing mice infused with GD2.BBζ CAR-T cells, wherein repeated 3-day dasatinib pulses slowed tumor growth and enhanced survival (Fig. 7, C and D, and fig. S11, C and D), and in Nalm6-GD2-bearing mice infused with drug-regulatable GD2.28ζ.ecDHFR CAR-T cells, wherein toggling CAR expression using a 1-week TMP/1-week OFF dosing schedule improved tumor control (Fig. 7, E and F).

20To confirm that rest mediated CAR-T cell exhaustion reversal in vivo, tumor bearing GD2.BBζ CAR-T treated mice were treated with dasatinib for 3 or 7 consecutive days starting at D16 or D12 post-engraftment (Fig. 7G), time points associated with onset of CAR-T cell exhaustion and failure to control tumor growth (fig. S11E). Tumor-infiltrating CAR-T cells (CAR-TIL) on D19 exhibited diminished inhibitory receptor expression, increased frequencies of memory-like cells, and augmented functionality (Fig. 7, H and I, and fig. S11, F and G), indicating that transient cessation of antigen-induced CAR signaling in vivo reverses hallmarks of exhaustion. Collectively, these results demonstrate that pharmacologic antagonism of TCR signaling kinases prevents or reverses CAR-T cell exhaustion and intermittent CAR-T cell rest results in superior antitumor responses in vivo.

21Chronic antigen stimulation induces T cell exhaustion, which is associated with a heritable epigenetic imprint distinct from effector and memory T cells. Therapeutic agents targeting the PD-1/PD-L1 axis can reinvigorate exhausted T cells, but do not reverse the exhaustion-associated epigenetic imprint. Recent studies have identified TOX, TOX2, and AP-1 family members as central regulators of T cell exhaustion that promote widespread transcriptional and epigenetic dysregulation, enabling new approaches to mitigate exhaustion, including enforced expression of c-Jun (S) or CRISPR-mediated deletion of TOX, TOX2, or NR4A family TFs. However, such modifications have not reversed the exhaustion-associated epigenetic imprint, leading many to suggest that the imprint is fixed.

22In this study, we modified a validated in vitro model of T cell exhaustion, wherein tonic CAR signaling induces hallmark phenotypic, functional, transcriptomic and epigenetic features of exhaustion within 11 days (HA.28ζ), to enable precise, drug-dependent control of CAR signaling. Consistent with murine models of viral infection wherein antigen clearance induces T cell memory rather than exhaustion, early cessation of CAR signaling (RestedD7-11) redirected T cell differentiation away from exhaustion and toward a memory-like state. When inhibition of CAR signaling was delayed until D11, after cells had already acquired hallmark features of T cell exhaustion, we observed impressive functional reinvigoration associated with global phenotypic, transcriptomic and epigenetic reprogramming. Similar results were obtained using the tyrosine kinase inhibitor dasatinib to inhibit CAR signaling, where functional reinvigoration was observed even in CAR-T cells subjected to prolonged antigen-independent tonic signaling or antigen-induced signaling in vivo.

23Several groups have sought to identify subsets of exhausted T cells with capacity for reversal of the exhaustion program, leading to the discovery of “progenitor exhausted” T cells, which express the stemness transcription factor TCF1 (gene name Tcf7) and exhibit increased accessibility at the Tcf7 locus. Progenitor exhausted cells exhibit greater reprogrammability and are apparently essential for the proliferative burst following PD-1 blockade. One model to explain our findings posits that cessation of tonic CAR signaling induces preferential expansion of progenitor exhausted T cells. Consistent with this, rested CAR-T cell populations exhibited increased accessibility at the Tcf7 locus, enriched Tcf7 binding motifs within accessible regions of the genome, and increased frequency of TCF1+ cells compared to exhausted CAR-T cells. However, TCF1+ cells in reinvigorated dasatinib-treated groups did not co-express PD-1 or other immune checkpoint receptors, a canonical feature of progenitor exhausted T cells. Further, clonotypic analyses demonstrated similarly high levels of TCR diversity in exhausted and rested cell populations, indicating that transcriptional and epigenetic alterations induced by rest in this model system did not involve preferential expansion of a small subset of clones.

24An alternative model that is most consistent with the data presented here posits that exhausted cell populations that have acquired the hallmark epigenetic imprint retain the capacity for epigenetic remodeling to resemble healthy, non-tonically signaling CAR-T cells. Results demonstrating that tazemetostat, an EZH2 inhibitor, prevents complete functional reinvigoration induced by rest in this model system are consistent with epigenetic remodeling rather than enrichment of progenitor exhausted cells. Future studies are warranted to better define the specific cell populations that undergo epigenetic remodeling following T cell rest and identify the precise role of EZH2 in remodeling of the exhaustion-associated epigenome.

25Irrespective of mechanism, these results demonstrate that cessation of CAR signaling augments function in cells transitioning to exhaustion and in those already endowed with hallmark features of exhaustion, and is distinct from that which is induced by PD-1/PD-L1 blockade. Further, dasatinib promoted T cell memory during ex vivo expansion, suggesting that incorporation of dasatinib during manufacturing of clinical CAR-T cell products could enhance efficacy following adoptive transfer, which is consistent with studies showing that ex vivo kinase inhibitor treatment improves T cell functionality.

26Our work also demonstrates that intermittent CAR signaling in vivo can prevent or reverse exhaustion and thereby enhance anti-tumor responses in liquid and solid tumor models independent of the CAR costimulatory domain (CD28 versus 4-1BB) or the propensity for tonic CAR signaling (CD19 binder versus GD2 binder). The findings raise the prospect that regulatable CAR platforms developed to mitigate CAR-mediated toxicity, including the DD-CAR system described here, may also enhance CAR-T cell efficacy as a result of temporal control of CAR-T cell signaling. Consistent with this, a recent study testing a regulatable CD19-targeting CAR demonstrated that CAR-T cells provided a longer rest phase exhibited superior antigen-induced expansion compared to those that received rest for a shorter period. Additional studies are needed to determine whether this approach can be universally applied to all CARs and whether remodeling of the exhaustion-associated epigenome is the mechanism by which rest augments functionality in non-tonic signaling CAR-expressing T cells in vivo. Of note, the observation that intermittent rest augments functionality is arguably paradoxical, since CAR-T cell inactivation would be expected to provide periods of unopposed tumor growth and thereby reduce efficacy. Ultimately, maneuvers designed to induce “rest” in the context of cancer immunotherapy are likely to be more effective if approaches are developed that enable phased periods of rest to a portion of the T cell population rather than resting the entire population en masse, and thereby maintaining consistent anti-tumor immune pressure.

27The findings presented here also raise the prospect that therapies designed to transiently inhibit TCR signaling might enhance functionality of exhausted, non-engineered T cell populations. This hypothesis has been tested to a limited extent in murine models of chronic virus or cancer, whereby removal of antigen failed to reinvigorate exhausted T cells. Discrepancies between our observations and those in murine models could be explained by intrinsic differences in murine and human exhausted T cells’ potential for functional reinvigoration. 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. Similarly, an immunomodulatory effect of dasatinib on T cells has been associated with improved anti-tumor immunity, and BRAF/MEK inhibition in patients with melanoma leads to upregulation of TCF7 and expansion of melanoma-specific TIL. Collectively, these observations suggest that transient cessation of TCR signaling could provide a widely applicable but underappreciated approach to enhance functionality in populations of exhausted human T cells; however, additional studies are needed to more fully define the effects of rest on non-engineered, exhausted T cells.

28In summary, we demonstrate that transient cessation of CAR signaling can restore functionality and induce epigenetic reprogramming in exhausted human CAR-T cell populations. These results suggest that CAR-T cell therapeutics designed to incorporate periods of rest may exhibit superior efficacy compared to constitutive platforms, and raise the prospect that targeting of proximal TCR/CAR signaling kinases may represent an immunotherapeutic strategy for mitigating T cell exhaustion.