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Early expansion and long-term persistence predict efficacy of chimeric antigen receptor T cells (CARTs)1–7, but mechanisms governing effector versus memory CART differentiation and whether asymmetric cell division induces differential fates in human CARTs remain unclear. Here we show that target-induced proximity labelling enables isolation of first-division proximal-daughter and distal-daughter CD8 CARTs that asymmetrically distribute their surface proteome and transcriptome, resulting in divergent fates. Target-engaged CARs remain on proximal daughters, which inherit a surface proteome resembling activated-undivided CARTs, whereas the endogenous T cell receptor and CD8 enrich on distal daughters, whose surface proteome resembles resting CARTs, correlating with glycolytic and oxidative metabolism, respectively. Despite memory-precursor phenotype and in vivo longevity, distal daughters demonstrate transient potent cytolytic activity similar to proximal daughters, uncovering an effector-like state in distal daughters destined to become memory CARTs. Both partitioning of pre-existing transcripts and changes in RNA velocity contribute to asymmetry of fate-determining factors, resulting in diametrically opposed transcriptional trajectories. Independent of naive, memory or effector surface immunophenotype, proximal-daughter CARTs use core sets of transcription factors known to support proliferation and effector function. Conversely, transcription factors enriched in distal daughters restrain differentiation and promote longevity, evidenced by diminished long-term in vivo persistence and function of distal-daughter CARTs after IKZF1disruption. These studies establish asymmetric cell division as a framework for understanding mechanisms of CART differentiation and improving therapeutic outcomes.

Engineered T-cell therapies have demonstrated impressive clinical responses in patients with hematologic malignancies. Despite this efficacy, many patients have a transient persistence of T cells, which can be correlated with transient clinical response. Translational data on T cells from pediatric cancer patients shows a progressive decline in chimeric antigen receptor (CAR) suitability with cumulative chemotherapy regardless of regimen. We investigated the effects of chemotherapy on surviving T cells in vitro, describing residual deficits unique to each agent including mitochondrial damage and metabolic alterations. In the case of cyclophosphamide but not doxorubicin or cytarabine, these effects could be reversed with N-acetylcysteine. Specifically, we observed that surviving T cells could be stimulated, expanded, and transduced with CARs with preserved short-term cytolytic function but at far lower numbers and with residual metabolic deficits. These data have implications for understanding the effects of chemotherapy on mature T cells later collected for adoptive cell therapy, as chemotherapy-exposed T cells may have lingering dysfunction that affects ex vivo adoptive cell therapy manufacturing techniques and, ultimately, clinical efficacy.

Engineered T-cell therapies have demonstrated impressive clinical responses in patients with hematologic malignancies. Despite this efficacy, many patients have a transient persistence of T cells, which can be correlated with transient clinical response. Translational data on T cells from pediatric cancer patients shows a progressive decline in chimeric antigen receptor (CAR) suitability with cumulative chemotherapy regardless of regimen. We investigated the effects of chemotherapy on surviving T cells in vitro, describing residual deficits unique to each agent including mitochondrial damage and metabolic alterations. In the case of cyclophosphamide but not doxorubicin or cytarabine, these effects could be reversed with N-acetylcysteine. Specifically, we observed that surviving T cells could be stimulated, expanded, and transduced with CARs with preserved short-term cytolytic function but at far lower numbers and with residual metabolic deficits. These data have implications for understanding the effects of chemotherapy on mature T cells later collected for adoptive cell therapy, as chemotherapy-exposed T cells may have lingering dysfunction that affects ex vivo adoptive cell therapy manufacturing techniques and, ultimately, clinical efficacy.

Cell-based therapeutics have considerable promise across diverse medical specialties; however, reliable human imaging of the distribution and trafficking of genetically engineered cells remains a challenge. We developed positron emission tomography (PET) probes based on the small-molecule antibiotic trimethoprim (TMP) that can be used to image the expression of the Escherichia colidihydrofolate reductase enzyme (eDHFR) and tested the ability of [18F]-TMP, a fluorine-18 probe, to image primary human chimeric antigen receptor (CAR) T cells expressing the PET reporter gene eDHFR, yellow fluorescent protein (YFP), and Renilla luciferase (rLuc). Engineered T cells showed an approximately 50-fold increased bioluminescent imaging signal and 10-fold increased [18F]-TMP uptake compared to controls in vitro. eDHFR-expressing anti-GD2 CAR T cells were then injected into mice bearing control GD2−and GD2+tumors. PET/computed tomography (CT) images acquired on days 7 and 13 demonstrated early residency of CAR T cells in the spleen followed by on-target redistribution to the GD2+tumors. This was corroborated by autoradiography and anti-human CD8 immunohistochemistry. We found a high sensitivity of detection for identifying tumor-infiltrating CD8 CAR T cells, ∼11,000 cells per mm3. These data suggest that the [18F]-TMP/eDHFR PET pair offers important advantages that could better allow investigators to monitor immune cell trafficking to tumors in patients.

Macrophages enforce antitumor immunity by engulfing and killing tumor cells. Although these functions are determined by a balance of stimulatory and inhibitory signals, the role of macrophage metabolism is unknown. Here, we study the capacity of macrophages to circumvent inhibitory activity mediated by CD47 on cancer cells. We show that stimulation with a CpG oligodeoxynucleotide, a Toll-like receptor 9 agonist, evokes changes in the central carbon metabolism of macrophages that enable antitumor activity, including engulfment of CD47+cancer cells. CpG activation engenders a metabolic state that requires fatty acid oxidation and shunting of tricarboxylic acid cycle intermediates for de novo lipid biosynthesis. This integration of metabolic inputs is underpinned by carnitine palmitoyltransferase 1A and adenosine tri-phosphate citrate lyase, which, together, impart macrophages with antitumor potential capable of overcoming inhibitory CD47 on cancer cells. Our findings identify central carbon metabolism to be a novel determinant and potential therapeutic target for stimulating antitumor activity by macrophages.

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of advanced hematologic cancers, showing impressive response rates in pivotal trials. Despite this success, challenges in CAR T-cell adoption persist due to tumor resistance mechanisms, manufacturing failures, and limitations in CAR T-cell persistence and effector function. Strategies that promote T-cell persistence, proliferation, and cytotoxic activity are essential to improving long-term outcomes of CAR T therapy.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO–CD8+T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1–CD8+CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies.

Allogeneic natural killer (NK) cell adoptive transfer has shown the potential to induce remissions in relapsed or refractory leukemias and lymphomas, but strategies to enhance NK cell survival and function are needed to improve clinical efficacy. Here, we demonstrated that NK cells cultured ex vivo with interleukin-15 (IL-15) and nicotinamide (NAM) exhibited stable induction of l-selectin (CD62L), a lymphocyte adhesion molecule important for lymph node homing. High frequencies of CD62L were associated with elevated transcription factor forkhead box O1 (FOXO1), and NAM promoted the stability of FOXO1 by preventing proteasomal degradation. NK cells cultured with NAM exhibited metabolic changes associated with elevated glucose flux and protection against oxidative stress. NK cells incubated with NAM also displayed enhanced cytotoxicity and inflammatory cytokine production and preferentially persisted in xenogeneic adoptive transfer experiments. We also conducted a first-in-human phase 1 clinical trial testing adoptive transfer of NK cells expanded ex vivo with IL-15 and NAM (GDA-201) combined with monoclonal antibodies in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) (NCT03019666). Cellular therapy with GDA-201 and rituximab was well tolerated and yielded an overall response rate of 74% in 19 patients with advanced NHL. Thirteen patients had a complete response, and 1 patient had a partial response. GDA-201 cells were detected for up to 14 days in blood, bone marrow, and tumor tissues and maintained a favorable metabolic profile. The safety and efficacy of GDA-201 in this study support further development as a cancer therapy.

•The levels of CD200 expressed by aberrant plasma cells in MM are sufficient to inhibit the activity of CAR T cells.•Compared with CD200R-knockout and dominant-negative approaches, a CD200R-CD28 switch optimally enhances CAR T-cell function in CD200+MM models.

We report on a search for gravitational-wave bursts in data from the three LIGO interferometric detectors during their third science run. The search targets sub-second bursts in the frequency range 100-1100 Hz for which no waveform model is assumed and has a sensitivity in terms of the root-sum-square(rss) strain amplitude of hrss~ 10?20Hz?1/2. No gravitational-wave signals were detected in the eight days of analysed data.

A Correction to this paper has been published: https://doi.org/10.1038/s41591-021-01248-2.

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringensneuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNAalone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNArestrains CAR T cell differentiation during ex vivoculture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNAhave superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNAshow enhanced antitumor efficacy. Arming CAR T cells with CpNAalso enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNAis an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Noncanonical NF-κB signaling enhances the survival of CAR T cells expressing the 4-1BB costimulatory domain.

Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Background:T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases.

Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic “drugs” for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control.

Hippen: University of Minnesota: Other: holds patents for the production and use of iTregs for clinical trials. Riley:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: Patents & Royalties: hold patents for the production and use of iTregs for clinical trials. June:Pfizer: Honoraria; Celldex: Consultancy, Equity Ownership; Tmunity: Equity Ownership, Other: Founder, stockholder ; Johnson & Johnson: Research Funding; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties. Kean:Juno Therapeutics, Inc: Research Funding. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

Thymic regulatory T cells (tTreg) or in vitroinduced Tregs (iTregs) adoptive transfer suppresses murine acute graft-versus-host disease (GVHD). Previously we demonstrated that plasmacytoid dendritic cell (pDC) indoleamine 2,3-dioxygenase (IDO) fosters the development of human iTregs through tryptophan (Trp) depletion and production of catabolites including kynurenines (Kyn). IDO is an immunosuppressive enzyme implicated in maternal-fetal acceptance, tumor immunity, and autoimmunity. IDO is highly up-regulated in the colon of mice and patients with GVHD; in rodents, the lack of IDO leads to increased colon pathology and accelerated lethality. Because pDCs are rare and difficult to purify and expand, we sought to determine whether an optimally suppressive iTreg could be generated using clinically amenable conditions that mimicked pDCs support (i.e. Low Trp+Kyn).

Aoyama: CHUGAI PHAMACEUTICAL CO.,LTD: Honoraria; Mochida Pharmaceutical Co.,Ltd: Honoraria; Kyowa Hakko Kirin Company,Limited: Honoraria. Milone:Novartis: Patents & Royalties, Research Funding. Miller:Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

Milone: Novartis: Patents & Royalties, Research Funding. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. PD-1 is an inhibitory receptor that attenuates TCR signaling. Its expression is inducible on T-cells, B-cells, NKT-cells, and activated monoytes. Interactions between PD-1 and its ligands deliver inhibitory signals that regulate T-cell activation, tolerance, and immune-mediated tissue damage.A blocking anti-PD-1 mAb given at the time of transplant markedly accelerated acute GVHD lethality in preclinical models via an interferon-gamma dependent mechanism. Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. PD-L2 expression was limited to hematopoietic cells, but hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. PD-L1-deficient hosts exhibit rapid mortality associated with increased gut T-cell homing and loss of intestinal epithelial integrity, increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation, hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter. During acute GVHD, PD-L1 was up-regulated on donor T-cells. Surprisingly, GVHD-induced lethality was significantly reduced in recipients of donor T cells devoid of PD-L1 and associated with reduced PD-L1-/-donor T-cell infiltration into lymphoid organs and gut, a retention of intestinal epithelial integrity, and a lower production of inflammatory cytokines. During GVHD, PD-L1-/-donor T cells showed increased apoptosis and reduced proliferation, as well as reduced glycolysis, glutaminolysis, and fatty acid metabolism. A role for PD-L1 in glucose-mediated acetyl-CoA production was seen, highlighting the important of glucose as an important carbon source in in alloreactive T cells undergoing clonal expansion. Further data support the hypothesis that the PD-1/PD-L1 pathway regulates T-T interaction. Together our studies indicate that PD-L1 expression that is upregulated on alloreactive donor T cells increases their survival and alters their metabolic pathway utilization in GVHD mice. In contrast to acute GVHD models, we have found that PD-1 pathway blockade can reduce chronic GVHD in a mouse model of multi-organ system disease in which one prominent component is bronchiolitis obliterans. This may occur via effects on T follicular regulatory or germinal center B cells. In summary, we have identified distinct consequences of PD-1/PD-L1 engagement in preclinical acute and chronic GVHD models: PD-1/PD-L1 interactions restrain acute GVHD but increase chronic GVHD. These findings illustrate the important but complex regulatory features of this pathway on a wide array of cell types. Our finding suggests PD-1 pathway modulation may provide unique opportunities for altering immune regulation post-transplant.

The PD-1/PD-L1 pathway plays an important role in regulation of alloimmune responses and in induction and maintenance of peripheral tolerance. Because GVHD is driven by donor T cells and PD-L1 expression can be markedly elevated on T cells during activation, we investigated the functional significance of PD-L1 expressed by donor T cells in regulating murine models of acute GVHD.