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Supplemental Table 1 Supplementary Figures 1-10

Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence.Significance:Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.This article is highlighted in the In This Issue feature, p. 627

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To “benchmark” exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8 + T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation–driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.

T cells are the major arm of the immune system responsible for controlling and regressing cancers. To identify genes limiting T cell function, we conducted genome-wide CRISPR knockout screens in human chimeric antigen receptor (CAR) T cells. Top hits were MED12 and CCNC , components of the Mediator kinase module. Targeted MED12 deletion enhanced antitumor activity and sustained the effector phenotype in CAR- and T cell receptor–engineered T cells, and inhibition of CDK8/19 kinase activity increased expansion of nonengineered T cells. MED12 -deficient T cells manifested increased core Meditator chromatin occupancy at transcriptionally active enhancers—most notably for STAT and AP-1 transcription factors—and increased IL2RA expression and interleukin-2 sensitivity. These results implicate Mediator in T cell effector programming and identify the kinase module as a target for enhancing potency of antitumor T cell responses.

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes limiting T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were MED12 and CCNC, components of the cyclin-dependent kinase (CDK) module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. MED12 or CCNC deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. Chemical inhibition of CDK8/19 kinase activity recapitulated some features of genetic loss of MED12, including increased T cell expansion. MED12 deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation at enhancers used by transcription factors playing a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in MED12 deficient T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK module as a target for enhancing the potency of anti-tumor T cell responses.One Sentence SummaryThe Mediator kinase module is a primary regulator of T cell differentiation, and genetic or small molecule-based inhibition of this module enhances effector T cell potency.

Introductory paragraphWhile CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity3–7; however, a truly safe and effective logic-gated CAR has remained elusive8. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3ζ domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumorsin vivowhile bypassing upstream signaling proteins such as CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineerLogic-gatedIntracellularNetworK(LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity9and fibrosis10. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.

Key Points Dasatinib potently and reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation. Dasatinib could be repurposed as a safety switch to mitigate CAR-mediated toxicity in patients.

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes that limit T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were components of the CDK8 kinase module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. CDK8 kinase module deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. CDK8 kinase module deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation most notably involving transcription factors that play a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in CDK8 kinase module deficient CAR-T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK8 kinase module as a target for enhancing the potency of anti-tumor T cell responses. Citation Format: Katherine A. Freitas, Julia A. Belk, Elena Sotillo, Bence Daniel, Katalin Sandor, Dorota Klysz, Vandon T. Duong, Peng Xu, Meena Malipatlolla, Evan W. Weber, Robbie G. Majzner, Howard Y. Chang, Ansuman T. Satpathy, Crystal Mackall. Enhanced effector activity of mediator CDK8 kinase module deficient CAR-T Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2822.

Chimeric Antigen Receptor (CAR) T cell therapy has resulted in remarkable clinical outcomes in the context of acute and chronic lymphoblastic leukemia, but remains unsuccessful in the treatment of solid tumors. One reason for this failure is thought to be T cell dysfunction or exhaustion promoted by suppressive soluble factors within the tumor microenvironment (TME). High extracellular levels of the immunosuppressive factor adenosine (Ado) are generated in the TME via breakdown of ATP by ecto-enzymes CD39 and CD73 expressed on tumor-infiltrating immune cells. Binding of extracellular Ado to its receptor A2a on T cells results in inhibition of proliferation and effector function. Interestingly, CD39 has recently been described as a surrogate marker of exhaustion on human CAR-T cells and non-engineered T cells. Therefore, we hypothesized that CD39 expression on exhausted CAR-T cells promotes dysfunction through generation of extracellular adenosine. Using an in vitro model of T cell exhaustion, whereby human T cells express a CAR that tonically signals in an antigen-independent manner (HA CAR), we demonstrate that exhausted HA CAR T cells actively hydrolyze extracellular ATP via their elevated expression of CD39 and CD73. Moreover, exhausted CD39+ CAR T cells upregulate several genes associated with a Treg phenotype at the mRNA and protein levels, suggesting that this cell population might be suppressive. To assess whether CD39+/CD73+ CAR T cells exhibit suppressive functions, we co-cultured them with non-exhausted CD19-CAR T cells. Indeed, proliferation and secretion of IL-2 by CD19 CAR T cells were diminished when they were co-cultured with exhausted CD39+ CAR T cells, and that this suppression is dependent on the A2a receptor. Using this knowledge, we used gene-editing and overexpression approaches to engineer CAR-T cells with resistance to suppressive adenosine signaling. In contrast to genetic deletion of CD39 or CD73, which did not alleviate CAR T cell dysfunction, genetic deletion of adenosine receptor A2aR in exhausted CAR T cells resulted in phenotypic changes and a modest improvement in tumor-specific killing. Further, ectopic overexpression of adenosine deaminase (ADA) in CAR T cells led to decreased exhaustion marker expression and significantly enhanced effector function. These data indicate that ADA overexpression is an innovative approach to increase the functionality of CAR T cells through avoidance of suppressive adenosine signaling, and provides proof-of-concept that metabolic engineering of CAR-T cells can pave the way for responses in patients with solid tumors. Citation Format: Dorota Klysz, Meena Malipatlolla, Katherine Freitas, Malek Bashti, Louai Labanieh, Peng Xu, Cecilia Ramello, Amaury Lerust, Hyatt Balke Want, Kaithlen Zen Pacheco, Evan W. Weber, Shabnum Patel, Steven Feldman, Elena Sotillo, Crystal L. Mackall. Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1362.

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poorin vivofunction, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how two methods which induce conventional T cell exhaustion – repetitive stimulation or expression of a tonic-signaling chimeric antigen receptor (CAR) – affect human Tregs. With each repetitive polyclonal stimulation Tregs progressively acquired an exhausted phenotype, and became less suppressivein vitro. Tregs expressing a tonic-signaling CAR rapidly acquired an exhausted phenotype and had major changes in their transcriptome and metabolism. Although tonic-signaling CAR-Tregs remained stable and suppressivein vitro, they lostin vivofunction, as tested in a model of xenogeneic graft-versus-host disease. The finding that human Tregs are susceptible to exhaustion has important implications for the design of Treg adoptive immunotherapy strategies.

Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. Significance: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence. This article is highlighted in the In This Issue feature, p. 627

SUMMARYT cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR-T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. “rest”) on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved anti-tumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.

The efficacy of chimeric antigen receptor (CAR) T cell therapy is limited by T cell exhaustion, whereby continuous CAR signaling induces hierarchical loss of effector function. Current methods to mitigate exhaustion in cancer, like immune checkpoint blockade, are not efficacious when combined with CAR T cells. Thus, new therapeutic approaches that target CAR T cell exhaustion are needed. To test the hypothesis that transient cessation of CAR signaling reverses CAR T cell exhaustion, we engineered human T cells to express a drug-regulatable CAR that demonstrates a high degree of antigen-independent tonic signaling and induces hallmarks of exhaustion within 10 days. Using this in vitro model of CAR T cell exhaustion, we discovered that transient CAR downregulation and concomitant cessation of tonic CAR signaling (i.e. “rest”) resulted in profound phenotypic changes, including decreased inhibitory receptor expression and increased memory-associated markers. Upon CAR re-expression and tumor challenge, rested CAR T cells exhibited improved killing, cytokine secretion, and sensitivity to antigen in vitro, as well as enhanced in vivo potency following adoptive transfer into tumor-bearing mice. Similar results were achieved via pharmacologic treatment with the Src kinase inhibitor dasatinib, which potently and reversibly inhibits CAR signaling. RNA- and ATAC-sequencing revealed that rest induces global transcriptional and epigenetic reprogramming, wherein rested CAR T cells closely resembled healthy, non-exhausted CAR T cells. Notably, epigenetic reprogramming and functional reinvigoration in rested cells were dependent on the activity of the histone methyltransferase EZH2, indicating that epigenetic alterations are required for functional reversal of CAR T cell exhaustion. Finally, providing multiple periods of rest in vivo by toggling CAR expression or pulsing with dasatinib reversed hallmarks of CAR T cell exhaustion and lead to more durable anti-tumor responses in xenograft murine models. This work demonstrates that transient cessation of CAR T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state. These results also raise the prospect that targeting proximal TCR/CAR signaling kinases might represent a novel immunotherapeutic strategy for mitigating exhaustion in human CAR T cells. Citation Format: Evan W. Weber, Crystal Mackall. Transient "rest" reinvigorates exhausted CAR T cells via epigenetic remodeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 61.

CAR-T cells rest to get back in the race Chimeric antigen receptor (CAR)–T cells, which are engineered to target specific tumor antigens, are increasingly used as an immunotherapy. CAR-T cells have shown promising results in patients, particularly in hematologic cancers, but their anticancer activity can be limited by the onset of exhaustion and the loss of effectiveness. Weber et al. characterized the phenotypic and epigenomic changes associated with CAR-T cell exhaustion caused by continuous activity and the beneficial effects of transient rest periods (see the Perspective by Mamonkin and Brenner). The authors tested different approaches for providing these rest periods, such as using the drug dasatinib to temporarily suppress T cell activity, which helped to prevent exhaustion and improved antitumor activity in mouse models. Science , this issue p. eaba1786 ; see also p. 34

SUMMARYCAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.

Leukocyte trafficking into the CNS is a prominent feature driving the immunopathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Blocking the recruitment of inflammatory leukocytes into the CNS represents an exploitable therapeutic target; however, the adhesion molecules that specifically regulate the step of leukocyte diapedesis into the CNS remain poorly understood. We report that CD99 is critical for lymphocyte transmigration without affecting adhesion in a human blood–brain barrier model. CD99 blockade in vivo ameliorated experimental autoimmune encephalomyelitis and decreased the accumulation of CNS inflammatory infiltrates, including dendritic cells, B cells, and CD4+ and CD8+ T cells. Anti-CD99 therapy was effective when administered after the onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest that it may serve as a novel therapeutic target for controlling neuroinflammation.

Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic.

We read with interest the article entitled, “High-affinity GD2-specific CAR T cells induce fatal encephalitis in a preclinical neuroblastoma model” by Richman and colleagues ([1][1]). In this report, the investigators generated a new chimeric antigen receptor (CAR) incorporating a mutated

Autoimmunity occurs through a break in tolerance, which is considered to be mediated by a failure to delete autoreactive immune cells. The central mechanism for deleting cells is controlled by the apoptotic machinery. Apoptosis occurs via two distinct pathways, an extrinsic pathway involving transduction of an apoptotic signal following aggregation of a death receptor, such as Fas, to its ligand, Fas ligand (FasL), and an intrinsic pathway that signals through the mitochondria and is regulated by the bcl-2 family. In the extrinsic pathway, binding of homotrimeric FasL to Fas facilitates recruitment of both Fas-associated death domain protein (FADD) and pro-caspase-8 leading to the activation of caspase-8 and subsequent degradative phase of apoptosis (1). This process may be inhibited by cellular FADD-like IL-1β-converting enzyme (FLICE)-inhibitory protein (cFLIP), which is also recruited by FADD and acts as an endogenous suppressor of the Fas pathway (1). Mice mutant for Fas (lpr) mice show normal postnatal development, but over time exhibit splenomegaly, lymphadenopathy, hypergammaglobulinemia, autoantibodies, glomerulonephritis, and the presence of double negative T-cells (CD3+CD4-CD8-B220+), with disease severity highly dependent upon strain background (2). The advent of conditional deletion of genes has yielded novel, unexpected, and controversial results regarding specific effects of loss of Fas within the various hematopoietic cell populations. While previous studies have extensively examined the role that Fas plays in lymphocytes and dendritic cells (3-5), few or no studies have directly examined the cell-intrinsic role of Fas in myeloid cells and its contribution to autoimmunity. Here, mice were generated that conditionally lack Fas only in the myeloid cell compartment (CreLysMFasflox/flox). These mice displayed a disruption in monocyte/macrophage and granulocyte homeostasis that led to altered activation of dendritic cells and lymphocytes in a paracrine fashion. Moreover, CreLysMFasflox/flox mice presented with splenomegaly, as well as autoantibodies and proinflammatory cytokines/chemokines. Furthermore, deposited immune complexes in kidneys and glomerulonephritis were observed in mice with myeloid cell-specific loss of Fas. Taken together, these data indicate that loss of Fas in myeloid cells is sufficient to induce an inflammatory phenotype in mice reminiscent of SLE-like disease, suggesting a role of specifically targeting Fas in its treatment.

Target antigen density has emerged as a major factor influencing the potency of CAR T cells. Our laboratory has demonstrated that the activity of numerous CARs is highly dependent on target antigen density (Walker et al., Mol Ther, 2017), and high complete response rates in a recent trial of CD22 CAR T cells for B-ALL were tempered by frequent relapses due to decreased CD22 antigen density on lymphoblasts (Fry et al., Nat Med, 2018). To assess if antigen density is also a determinant of CD19 CAR T cell therapeutic success, we analyzed CD19 antigen density from fifty pediatric B-ALL patients treated on a clinical trial of CD19-CD28ζ CAR T cells. We found that patients whose CD19 expression was below a threshold density (2000 molecules/lymphoblast) were significantly less likely to achieve a clinical response than those whose leukemia expressed higher levels of CD19. In order to further understand this limitation and how it may be overcome, we developed a model of variable CD19 antigen density B-ALL. After establishing a CD19 knockout of the B-ALL cell line NALM6, we used a lentivirus to reintroduce CD19 and then FACS sorted and single cell cloned to achieve a library of NALM6 clones with varying CD19 surface densities. CD19-CD28ζ CAR T cell activity was highly dependent on CD19 antigen density. We observed decreases in cytotoxicity, proliferation, and cytokine production by CD19 CAR T cells when encountering CD19-low cells, with an approximate threshold of 2,000 molecules of CD19 per lymphoblast, below which, cytokine production in response to tumor cells was nearly ablated. Given that a CD19-4-1BBζ CAR is FDA approved for children with B-ALL and adults with DLBCL, we wondered whether CARs incorporating this alternative costimulatory domain would have similar antigen density thresholds for activation. Surprisingly, CD19-4-1BBζ CAR T cells made even less cytokine, proliferated less, and had further diminished cytolytic capacity against CD19-low cells compared to CD19-CD28ζ CAR T cells. Analysis by western blot of protein lysates from CAR T cells stimulated with varying amounts of antigen demonstrated that CD19-CD28ζ CAR T cells had higher levels of downstream signals such as pERK than CD19-4-1BBζ CAR T cells at lower antigen densities. Accordingly, calcium flux after stimulation was also significantly higher in CD19-CD28ζ than CD19-4-1BBζ CAR T cells. In a xenograft model of CD19-low B-ALL, CD19-4-1BBζ CAR T cells demonstrated no anti-tumor activity, while CD19-CD28ζ CAR T cells eradicated CD19-low leukemia cells. Therefore, the choice of costimulatory domain in CAR T cells plays a major role in modulating activity against low antigen density tumors. CD28 costimulation endows high reactivity towards low antigen density tumors. We confirmed the generalizability of this finding using Her2 CAR T cells; Her2-CD28ζ CAR T cells cleared tumors in an orthotopic xenograft model of Her2-low osteosarcoma, while Her2-4-1BBζ CAR T cells had no effect. This finding has implications for CAR design for lymphoma and solid tumors, where antigen expression is more heterogeneous than B-ALL. To enhance the activity of CD19-4-1BBζ CAR T cells against CD19-low leukemia, we designed a CAR with two copies of intracellular zeta in the signaling domain (CD19-4-1BBζζ). T cells expressing this double-zeta CAR demonstrated enhanced cytotoxicity, proliferation, cytokine production, and pERK signaling in response to CD19-low cells compared to single-zeta CARs. Additionally, in a xenograft model, CD19-4-1BBζζ CAR T cells demonstrated enhanced activity against CD19-low leukemia compared to CD19-4-1BBζ CAR T cells, significantly extending survival. The addition of a third zeta domain (CD19-4-1BBζζζ) further enhanced the activity of CAR T cells. However, inclusion of multiple copies of the costimulatory domains did not improve function. In conclusion, CD19 antigen density is an important determinant of CAR T cell function and therapeutic response. CD19-CD28ζ CARs are more efficient at targeting CD19-low tumor cells than CD19-4-1BBζ CARs. The addition of multiple zeta domains to the CAR enhances its ability to target low antigen density tumors. This serves as proof of concept that rational redesign of CAR signaling endodomains can result in enhanced function against low antigen density tumors, an important step for extending the reach of these powerful therapeutics and overcoming a significant mechanism of tumor escape. Disclosures Lee: Juno: Consultancy.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell leukemia. However, application of CAR T cell therapy in solid tumors has been disappointing. We believe the development of T cell exhaustion, a phenomenon well described in chronic viral infection, is a major barrier to widespread success of CAR T cell therapy in other types of cancer. Therefore, understanding mechanisms of and engineering solutions to avoid the development of CAR T cell exhaustion is highly warranted. Here, we utilize a high affinity (HA) GD2-directed CAR model in which T cells undergo chronic activation via spontaneous clustering of surface CAR molecules. Tonically signaling HA-28Z CAR T cells demonstrate phenotypic and functional characteristics of exhaustion with high expression of inhibitory receptors (PD-1, TIM-3, LAG-3, and CD39), poor cytokine production, enhanced effector differentiation, and failure to eradicate disease upon transfer into tumor-bearing mice. To interrogate the transcriptional and epigenetic programs driving CAR T cell exhaustion, we performed total RNA-seq and ATAC-seq using HA-28Z or control CD19-28Z CAR T cells. The most significantly enriched transcription factor motifs in HA-specific open chromatin peaks belonged to the AP1 family, suggesting AP1 factors may play an integral role in mediating exhaustion. RNA-seq and western blot confirmed increased expression of AP1 factors in exhausted CAR T cells. Interestingly, exhausted HA-28Z CAR T cells demonstrate insufficient IL2 production, a gene classically regulated by AP1 Fos/cJun heterodimers, although Fos and cJun are present in CAR T cells. However, other potentially inhibitory AP1 family members (including JunB, BATF, ATF, and IRF family members) are overexpressed to a greater degree. We hypothesize that the relative balance of classical and inhibitory AP1 family members may contribute to exhaustion. To disrupt this balance, we constitutively overexpressed Fos and cJun in exhausted HA-28Z CAR T cells and observed that JUN+ CAR T cells demonstrated increased IL2 and IFNg production and reduced inhibitory receptor expression. Furthermore, adoptively transferred JUN-HA-28Z CAR T cells resulted in rapid tumor clearance and long term survival of NSG mice bearing GD2+ leukemia whereas control HA-28Z CAR T cells could not overcome tumor growth. Co-immunoprecipitation experiments show high levels of JunB/BATF3/IRF4 complexed with cJun in exhausted HA-28Z CAR T cells. Preliminary data using CRISPR-Cas9 gene disruption suggest that knocking out inhibitory AP1 factors in HA-28Z CAR T cells can also enhance functional activity. Together, our data highlight the AP1 family in regulating T cell dysfunction upon chronic antigen encounter. Finally, we demonstrate that AP1 factors can be engineered to produce superior T cells for adoptive cell therapy. Citation Format: Rachel C. Lynn, Evan W. Weber, David Gennert, Elena Sotillo, Robert Jones, Peng Xu, Ansuman Satpathy, Howard Y. Chang, Crystal L. Mackall. Engineering AP1 to combat CAR T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-112.

In patients with chronic viral infection or cancer, continuous antigen exposure results in T cell exhaustion, which is characterized by sustained co-expression of multiple inhibitory receptors (ex. PD-1, TIM-3, LAG-3) and a hierarchical loss of effector function. Human T cells expressing a high-affinity anti-GD2 chimeric antigen receptor (CAR, HA.28z) develop phenotypic and functional hallmarks of exhaustion due to clustering of surface CAR and tonic signaling in the absence of antigen. In the present study, we exploited the HA.28z CAR as a model to characterize human T cell exhaustion and assess its potential for reversibility. CyTOF analyses of dysfunctional HA.28z CAR T cells demonstrated a distinct phenotype in which canonical exhaustion markers, T cell differentiation markers, and effector transcription factors were differentially expressed compared to CD19.28z CAR T cells that do not tonically signal. To interrogate the reversibility of T cell exhaustion, we engineered a regulatable HA.28z CAR by fusing a destabilization domain to the C-terminus of the CAR that rapidly induces CAR protein degradation. Addition of a small molecule stabilizes the protein and results in CAR surface expression. Using this model, we modulated the duration and intensity of tonic signaling by precisely modulating CAR surface expression. We hypothesized that transient cessation of tonic signaling would allow exhausted CAR T cells to recover and regain effector function. Following onset of exhaustion, elimination of CAR surface expression for 4 days resulted in a profound reversal of the exhausted phenotype. CyTOF analyses of reinvigorated cells indicated enhanced T cell memory formation, diminished surface marker expression of multiple exhaustion markers (ex. PD-1, TIM-3, LAG-3, CTLA-4, CD39) and reduced expression of transcription factors T-bet and Blimp-1. Furthermore, RNA sequencing of exhausted and reinvigorated HA.28z CAR T cells revealed a global molecular reprogramming upon removal of tonic CAR signaling, suggesting that CAR T cell exhaustion is functionally reversible. Upon CAR re-expression and co-culture with tumor antigen, reinvigorated HA.28z CAR T cells exhibited augmented killing and cytokine secretion compared to exhausted cells that continuously expressed surface CAR during expansion. In vivo studies assessing CAR T cell exhaustion reversibility using this model are currently being evaluated. In summary, tuning CAR surface expression offers a novel strategy to augment both the safety and efficacy of CAR T cell therapy. Moreover, these studies suggest that transient “rest” in T cells experiencing chronic antigen stimulation may be one mechanism by which exhaustion is prevented or reversed. Citation Format: Evan W. Weber, Rachel C. Lynn, Meena Malipatlolla, Elena Sotillo, Peng Xu, Crystal L. Mackall. Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-111.

CD99-Like 2 (CD99L2) is a Type I glycoprotein expressed on leukocytes and endothelial cells as well as other cell types. It is related to CD99, although it shows only 38% sequence identity. CD99L2 has been shown to play a role in leukocyte extravasation in mice under various inflammatory conditions using anti-CD99L2 antibodies and, in one case by targeted deletion of CD99L2. We report here studies on an independently made CD99L2 “knockout mouse” that extend our knowledge of the role of CD99L2 in inflammation. CD99L2 deficiency did not affect the total or relative numbers of circulating leukocyte subsets, red blood cells, or platelets. Neither did CD99L2 deficiency affect the expression of ICAM-1, PECAM, or CD99 on endothelial cells. Mice lacking CD99L2 had a defective inflammatory response in the thioglycollate peritonitis model with a greater than 80% block in neutrophil infiltration and a nearly complete block in monocyte emigration into the peritoneal cavity measured 16 hours after the inflammatory challenge. The mice will be a useful resource to study the role of CD99L2 in various acute and chronic inflammatory diseases.

OBJECTIVE: The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. METHODS: Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. RESULTS: Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. CONCLUSION: These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA., The authors are grateful for the assistance of staff of the Washington University School of Medicine Center for Musculoskeletal Biology and Medicine and the Northwestern University Feinberg School of Medicine Cell Imaging Core Facility and the Methodology and Data Management Core.

Weber et al. identify TRPC6 as the calcium channel mediating the transient increase in endothelial cytosolic free calcium concentration required for transendothelial migration of leukocytes during the inflammatory response., Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca2+]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca2+]i regulates TEM and the channels mediating this ↑[Ca2+]i are unknown. Buffering ↑[Ca2+]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca2+]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca2+ channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca2+]i required for TEM at a step downstream of PECAM homophilic interactions.

The DICER1 syndrome, also known as the pleuropulmonary blastoma familial tumor dysplasia syndrome (PPB-FTDS) (OMIM #601200), is a recently described entity comprising a number of rare to ultra-rare tumors arising mainly in childhood or adolescence. The most frequent and characteristic disorders are pleuropulmonary blastoma, cystic nephroma and ovarian Sertoli-Leydig cell tumors. Some aspects of the syndrome were identified in the 1970s and 1990s, but discovery in 2009 by Hill et al of heterozygous disease-associated germ-line DICER1 mutations in affected kindred brought the syndrome into focus. Several studies since 2009 have extended the phenotypes to include more common conditions such as multinodular goiter and Wilms tumor, as well as much rarer entities such as cervical embryonal rhabdomyosarcoma, pineoblastoma and pituitary blastoma. The critical molecular defect appears to be impairment of DICER1's RNase III endonuclease function, which normally would cleave precursor microRNAs to their final mature length. These microRNAs function by targeted silencing and/or degradation of specific messenger RNAs. DICER1 may be considered an unusual type of tumor suppressor gene, in that the first inherited “hit” usually cripples one allele completely, whereas a second somatic “hit” is nearly always limited to the RNase III domains (and is in fact often even more focused on the metal-binding domains of RNase IIIb). These second hits are most commonly a single base substitution leading to an amino acid change, which functionally impairs the protein without overall protein loss. In this presentation I will summarize the current knowledge on the role of DICER1 mutations in cancer and will describe the edges of the known associated phenotypes. Citation Format: William D. Foulkes, Mona Wu, Leanne De Kock, Leora Witkowski, Nelly Sabbaghian, Evan Weber, Nancy Hamel, John R. Priest. DICER1: From ontogenesis to oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA03. doi:10.1158/1538-7445.CANSUSC14-IA03